research article cross-platform evaluation of commercial real-time sybr green rt-pcr...

Research ArticleCross-Platform Evaluation of Commercial Real-TimeSYBR Green RT-PCR Kits for Sensitive and Rapid Detectionof European Bat Lyssavirus Type 1

Evelyne Picard-Meyer Carine Peytavin de Garam Jean Luc Schereffer Clotilde MarchalEmmanuelle Robardet and Florence Cliquet

Anses Laboratory for Rabies and Wildlife WHO Collaborating Centre for Research and Management in Zoonoses ControlOIE Reference Laboratory for Rabies European Union Reference Laboratory for Rabies European Union Reference Laboratoryfor Rabies Serology Technopole Agricole et Veterinaire CS 40009 54220 Malzeville France

Correspondence should be addressed to Evelyne Picard-Meyer evelynepicard-meyeransesfr

Received 3 October 2014 Accepted 12 January 2015

Academic Editor Cheng-Feng Qin

Copyright copy 2015 Evelyne Picard-Meyer et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

This study evaluates the performance of five two-step SYBR Green RT-qPCR kits and five one-step SYBR Green qRT-PCRkits using real-time PCR assays Two real-time thermocyclers showing different throughput capacities were used The analysedperformance evaluation criteria included the generation of standard curve reaction efficiency analytical sensitivity intra- andinterassay repeatability as well as the costs and the practicability of kits and thermocycling times We found that the optimisedone-step PCR assays had a higher detection sensitivity than the optimised two-step assays regardless of the machine used while nodifference was detected in reaction efficiency R2 values and intra- and interreproducibility between the two methods The limit ofdetection at the 95 confidence level varied between 15 to 981 copies120583L and 41 to 171 for one-step kits and two-step kits respectivelyOf the ten kits tested the most efficient kit was the Quantitect SYBR Green qRT-PCR with a limit of detection at 95 of confidenceof 20 and 22 copies120583L on the thermocyclers Rotor gene Q MDx and MX3005P respectively The study demonstrated the pivotalinfluence of the thermocycler on PCR performance for the detection of rabies RNA as well as that of the master mixes

1 Introduction

Rabies which is caused by all members of the genus Lyssavi-rus family Rhabdoviridae is a viral encephalitis that isfound on every continent except Antarctica The Lyssavirusgenus is divided into 14 species the majority of which arepredominantly associated with infection in bats [1] Bats arenatural reservoirs for all lyssaviruses and are considered torepresent the ancestral host for the lyssaviruses [2] In Europebat rabies is caused by four lyssaviruses namely Europeanbat Lyssavirus type 1 (EBLV-1) European bat Lyssavirus type2 (EBLV-2) Bokeloh bat Lyssavirus (BBLV) and Lleida batLyssavirus (LLEBV) Between 1977 and 2013 more than 1000cases of bat rabieswere reported [3] with themajority (gt97)being attributed to EBLV-1

Rabies diagnosis based upon clinical presentation orgross pathognomonic lesions is unreliable because signsof the disease are not characteristic and may vary greatlyfrom one animal to another Therefore diagnosis in ani-mals relies on postmortem laboratory findings The WHOldquogold standardrdquo test for rabies diagnosis in animals is thefluorescent antibody test (FAT) in which FITC anti-rabiesvirus antibody is applied to selected regions of the braincollected postmortem [4] Evidence of a rabies infection canbe demonstrated through the detection of antigen (FAT)infectious virus (rabies tissue culture infection test (RTCIT)mouse inoculation test (MIT)) or viral RNA (nucleic acidamplification tests) these conventional OIE diagnostic testsbeing entirely dependent on the nature and quality of thesample supplied With the advent of molecular biology since

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 839518 18 pageshttpdxdoiorg1011552015839518

2 BioMed Research International

the 1980s and due to the immense versatility and sensitivityof PCR diagnostic methods based on PCR technology havebeen extensively applied to many infectious diseases [5]To date real-time PCR is the state-of-the-art technologyfor the quantification of nucleic acids Various nucleic aciddetection-based assays have thus been published for thedetection of Lyssavirus fragments using conventional gel-based PCR NASBA or real-time PCR [6] In Europe mostlaboratories working on rabies employ conventional andorreal-time PCR [7] due to their high specificity and sensitivityThe conventional methods are reported to be very sensitivewhen using a heminested RT-PCR [8] and compared to hem-inested RT-PCR real-time PCR is shown to be about 100ndash1000-fold more sensitive [9] However due to this high PCRsensitivity which engenders a risk of sample cross contamina-tion and therefore a risk of potential false positive results RT-PCR is not recommended by the World Health Organizationfor routine postmortem diagnosis of rabies in animals [4]

Real-time RT-PCR which provides rapid sensitive andspecific detection of rabies virus decreases the risk of samplecontamination Various real-time assays have thus beendescribed since 2004 [10] for the rapid detection of RABVor other Lyssavirus species [11 12] using the one-step method[13 14] or the two-step method [12 15 16] Two systems ofreal-time assays are widely used for the detection of RABVand lyssaviruses the SYBR Green real-time RT-PCR andthe TaqMan real-time RT-PCR which is to date the mostcommon technique implemented in the laboratories workingon rabies [17]

As real-time PCR assays have become routine in diagnos-tic laboratories and because of their prominence there hasbeen rapid growth of commercially available reagent systemsand detection platforms which greatly increase the optionsavailable to laboratories In this study we evaluated severalcommercial master mixes in order to identify those whichproduce the best results using one-step and two-step real-time RT-PCR for the determination of EBLV-1 Five differentsystems for the generation of cDNA five two-step PCR kitsand five one-step qRT-PCR kits were comparedThe analysedperformance evaluation criteria included the generation ofa standard curve reaction efficiency analytical sensitivityintra- and interassay repeatability as well as the costs and thepracticability of kits and thermocycling times The goal ofthis study was also to determine if the one-step and two-stepsystems had the same reaction efficiencies for the detectionof EBLV-1 and if one system was more sensitive than anotherusing two real-time thermocyclers

2 Materials and Methods

21 Rabies Virus Isolates and RNA Extraction The Europeanbat Lyssavirus type 1 virus (EBLV-1) was selected for this studyfor the generation of in vitroRNA transcriptThe virus EBLV-1 (ANSES strain numbered 05-09) was isolated in France in2000 from a rabid bat (Eptesicus serotinus) The isolate wasmouse passaged before RNA extraction

Viral RNAwas extracted from 200120583L of 10mouse braintissue suspension using the Iprep Pure Link Virus kit (Invit-rogen France) according to the manufacturerrsquos instructions

The RNA concentration was determined spectrophotometri-cally and the RNA was stored at minus70∘C until use

22 Preparation of In Vitro RNA Transcripts Briefly a one-step RT-PCR amplification was performed as previouslydescribed [8] on 5 120583L of extracted RNA (ie 250ndash500 ng)with specific rabies primers (JW12 (forward) 51015840-ATGTAA-CACCYCTACAATG and JW6 (reverse) 51015840-CARTTVGCR-CACATYTTRTG) The 606 bp PCR products were purifiedand inserted into a pGEM T easy vector (Promega France)according to the manufacturerrsquos instructions Recombinantplasmids were purified using the PerfectPrep EndoFreePlasmid Maxi Kit (5Prime France) and then concentratedusing a step of sodium acetateethanol precipitation and lin-earised with the PstI restriction enzyme (Promega France)The clones of PCR products were bidirectionally sequencedwith sense and antisense primers T7 and SP6 by BeckmanCoulter Genomics (Takeley United Kingdom) to confirm thepresence of the target insert

In vitro transcription was performed using T7 RNApolymerase according to the manufacturerrsquos instructions(MaxiScript SP6T7 kit Ambion France)The resulting RNAtranscripts were purified as recommended by the manufac-turer using 2120583L of DNAse I during 15min at 37∘C followedby a final purification step using the RNeasymini kit (QiagenFrance) The RNA concentration was determined by usingthe Qubit Fluorometer (Invitrogen France) RNA transcriptswere aliquoted to 107 copies120583L and then stored at minus70∘CTheRNAmolecule copy numberwas calculated following themanufacturerrsquos instructions (MaxiScript SP6T7 kit AmbionFrance) using the following formula 119884 molecules120583L =(119883 g120583L RNA[transcript length in nucleotides times 330] times6022sdot1023) The estimated number of copies was of 268 sdot 1011for EBLV-1 RNA transcript

The quality of RNA transcript was checked on serialtenfold dilutions (106 to 1 copie120583L) through measurementsof efficiency of amplification using a two-step SYBR GreenRT-PCR [12] PCR efficiency was of 99

23 Real-Time RT-PCR Assays

231 Primers Used Universal pan Lyssavirus primers wereselected for this study Two questionnaires respectivelyundertaken in 2011-2012 by the EURL laboratory havedemonstrated that 13 out of 21 National Reference Laborato-ries for Rabies (NRLs) used primers JW12 and N165-146 pre-viously described by Wakeley et al [18] These specific rabiesprimers (JW12 (forward) ATGTAACACCYCTACAATGand N165-146 (reverse) GCAGGGTAYTTRTACTCATA)were designed to detect the rabies virus N gene of all knownlyssaviruses [12]

232 Two-Step RT-qPCR Assays

(1) Commercial Master Mix Kits

(a) Reverse Transcription Kits (Table 1(a)) Five differentreverse transcription (RT) systems provided by five differentcompanies and five different qPCR kits provided by the same

BioMed Research International 3

Table 1 Characteristics of the commercial master mix kits used In (a) the reactions mix and thermocycling conditions for the five RT kitsare detailed in (b) the reactions mix and thermocycling conditions for the five qPCR SybrGreen kits (two-step) and in (c) the reactions Mixand thermocycling conditions for the five qRT-PCR SybrGreen kits (one-step) used in this study

(a) Reactions mix and thermocycling conditions for the five RT kits

Manufacturer Reversetranscription kit RTase and primers Thermocycling

conditions Reaction mixlowastlowast

Bio-Rad Iscript select cDNAsynthesis kit

M-MLV RTlowastRandom primer

5min-25∘C30min-42∘C5min-85∘C

4 120583L Iscript Mix (5x) + 2120583L Random primer +1 120583L Iscript RTase + 8 120583L RNase free water

QiagenQuantiTectReverse

Transcription Kit

Quantiscript RTaseMix OligodT

+ random primer


2120583L gDNAWipeout Buffer (7x) + 4 120583LQuantiscript RT Buffer + 1120583L QuantiscriptRTase + 1 120583L RT primer + 7 120583L RNase free

water

FermentasMaxima HMinusFirst Strand cDNA

Synthesis Kit


5min-65∘C10min-25∘C30min-50∘C5min-85∘C

4120583L RT Buffer (5x) + 1 120583L Random primer +1 120583L dNTP (10mM) + 1 120583L Maxima HMinus

Ez Mix + 8 120583L RNase free water

Invitrogen SuperScript ViloMaster Mix



4120583L SuperScript Vilo MasterMix (5x) + 11 120583LRNase free water

Quanta qScript cDNASynthesis Kit

M-MLV RTlowastMix OligodT

+ random primer


4 120583L qScript Reaction Mix (5x) + 1 120583L qScriptRTase + 10 120583L RNase free water

lowastMoloney Murine Leukemia ViruslowastlowastTotal volume of reaction is 20120583L containing 5120583L of RNA sample and 15 120583L of reaction mix All RT assays were performed on the Mastercycler Eppendorfthermal cyclerRTase reverse transcriptase

(b) Reactions mix and thermocycling conditions for the five qPCR SybrGreen kits (two-step)

Manufacturer qPCRSybrGreen kit Thermocycling conditions Reaction mixlowast

Bio-RadSsoFastEvaGreenSupermix

30 sec-95∘C45 cycles of 30 sec-95∘C 30 sec-55∘C 30 sec-72∘C

Melt Curve (60∘C to 95∘C)

Total volume of 20 120583L containing 2 120583L of cDNA and18 120583L of a reaction mix (1x Sso Fast master mix035 120583M [05 120583M] each of pan-Lyssavirus primers)

QiagenQuantiTectSYBR GreenPCR Kit

15min-95∘C45 cycles of 30 sec-95∘C 30 sec-55∘C 30 sec-72∘C


Total volume of 25 120583L containing 2 120583L of cDNA and23 120583L of reaction mix (1x Quantitect Master mix05 120583M [06 120583M] each of pan-Lyssavirus primers)

FermentasMaxima SYBRGreen qPCRMaster Mix



Total volume of 25 120583L containing 2 120583L of cDNA and23 120583L of reaction mix (1x Maxima SYBR Green mastermix 04 120583M [1 120583M] each of pan-Lyssavirus primers)

Invitrogen

Express SYBRGreenER qPCR

SupermixUniversal



Total volume of 20 120583L containing 2 120583L of cDNA and18 120583L of reaction mix (1x Express SYBR GreenERSupermix 04 120583M [2 120583M] each of pan-Lyssavirus

primers) [50 nM of ROX dye]

Quanta Perfecta SYBRGreen SuperMix



Total volume of 25 120583L containing 2 120583L of cDNA and23120583L of reaction mix (1x Perfecta SYBR GreenSupermix 03 120583M [1 120583M] each of pan-Lyssavirus

primers)lowastThe PCR reaction protocol corresponds to PCR assays performed on the Rotor Gene Q MDx The concentrations of primers used on the real timethermocycler Mx3005P are shown in brackets [ ]

(c) Reactions mix and thermocycling conditions for the five qRT-PCR SYBR Green kits (one-step)

Manufacturer qRT-PCRSybrGreen kit Thermocycling conditions Reaction mixlowast

Qiagen QuantiTect SYBRGreen RT-PCR Kit

30min-50∘C 15min-95∘C45 cycles of 30 sec-95∘C 30 sec-55∘C 30 sec-72∘C


1x QuantiTect SYBR Green master mix1 Quantitect RT mix 06 120583M [2120583M]

each of pan-Lyssavirus primers


(c) Continued


QiagenRotor Gene-1 stepRotor-Gene SYBRGreen RT-PCR Kit



1x Rotor-Gene master mix 1Rotor-Gene RT mix 1 120583M [2 120583M] each of

pan-Lyssavirus primers

Fermentas Verso 1-stepQRT-PCR Kit



1x Verso SYBR Green master mix 5 RTenhancer 1 Verso enzyme mix 02 120583M[03 120583M] each of pan-Lyssavirus primers

Invitrogen

SuperScript IIIPlatinum SYBRGreen One-StepqRT-PCR Kit



1x SuperScript III Platinium master mix2 enzyme mix 03 120583M [05120583M] each of


QuantaqScript One-StepSYBR GreenRT-qPCR Kit



1x qScript master mix 2 qScript enzymemix 04 120583M [02 120583M] each of

pan-Lyssavirus primerslowastTotal volume of reaction is 25 120583L containing 2120583L of RNA sample and 23120583L of reactionmixThe PCR reaction protocol corresponds to PCR assays performedon the Rotor Gene Q MDx The concentrations of primers used on the real time thermocycler Mx3005P are shown in brackets [ ]

five suppliers were tested with the two-step method Thefollowing RT kits were compared Iscript (IScript cDNA syn-thesis kit Bio-Rad France) QuantiTect (Quantitect Reversetranscription kit Qiagen France) Maxima (Maxima HMinus First Strand cDNA synthesis kit Fermentas France)SS Vilo (Superscript Vilo Master Mix Invitrogen France)and qScript (qScript cDNA Synthesis kit Quanta France)

(b) Two-Step SYBR Green PCR Kits (Table 1(b)) Differenttwo-step qPCR kits were tested SsoFast (SsoFast EvagreenSuper mix kit Bio-Rad France) QuantiTect (QuantiTectSYBR Green PCR kit Qiagen France) Maxima (MaximaSYBR Green qPCR master mix Fermentas France) Express(Express SYBR GreenER qPCR Supermix universal Invitro-gen France) and Perfecta (Perfecta SYBR Green SupermixQuanta France)

(2) Two-Step RT-qPCR Analysis Seven 1 10 serial dilutions(ie 106 to 1 copie120583L) of the in vitro EBLV-1 RNA transcriptwere prepared in RNAse free water then subjected to reversetranscription using the five different RT master mixes AllRT kits were optimised with regard to the time and the tem-perature of incubation (Table 1(a)) Complementary cDNAswere generated from 5120583L of each dilution of EBLV-1 RNArandomprimer oligo dT or amix of oligo dTrandomprimerincluded in each tested kit according to the recommendationsof themanufacturersThe cycling conditions and the reactionprotocol for each tested RT kit are detailed in Table 1(a)Ther-mocycling was performed on the Mastercycler Eppendorfthermal cycler (Eppendorf France)

Real-time PCRs were run on the thermocyclers RotorGene QMDx (Qiagen France) and Mx3005P (Agilent Tech-nologies France) The concentrations of the reverse (N165-146) and forward (JW12) primers were optimised for eachtested kit At least three different concentrations were exam-ined that is the one recommended by the manufacturerone below and one above the recommended primer concen-tration using the qPCR conditions described in Table 1(b)The optimised concentration of primers is recorded for each

tested qPCR kits in Table 1(b) Reactions were performed intriplicate in a total volume of 20 120583L or 25 120583L following themanufacturerrsquos instructions (Table 1(b))

The following thermal profile was used PCR initialactivation step at 95∘C for 30 sec (SsoFast) 15min (Quan-tiTect) 10min (Maxima) 2min (Express) or 3min (Per-fecta) according to themanufacturerrsquos recommendationsThecycler conditions performed as previously described byHay-man et al [19] were the same for all assays 45 cycles of three-step cycling consisting of denaturation at 94∘C for 30 secannealing at 55∘C for 30 sec and extension at 72∘C for 30 sec

233 One-Step qRT-PCR Assays

(1) Commercial qRT-PCR Kits (Table 1(c)) Five commer-cial one-step kits provided by four suppliers were testedQuantiTect-1 step (QuantiTect SYBR Green RT-PCR kitQiagen France) RotorGene-1 step (Rotor-Gene SYBRGreenRT-PCR kit Qiagen France) Verso-1 step (Verso SYBRGreen one-step qRT-PCR kit Fermentas France) SSIIIPlatinium-1 step (SuperScript III Platinium SYBR GreenOne-step qRT-PCR kit Invitrogen France) and qScript-1step (qScript One step SYBR Green QRT-PCR kit QuantaFrance)

(2) One-Step qRT-PCR Analysis To compare the five mastermixes the assays were performed in triplicate using seventenfold dilutions (ie 106 to 1 copies120583L) of EBLV-1 RNAPCR was performed using a total volume of 25 120583L containing2 120583L of serial dilutions of EBLV-1 RNA and the master mixcomponents (119881 = 23 120583L) from each representative kit whichwere prepared according to the respective manufacturerrsquosinstructions (Table 1(c)) The concentrations of the reverse(N165-146) and forward (JW12) primers were optimised foreach tested kit (Table 1(c)) At least three different con-centrations were examined the one recommended by themanufacturer one below and one above the recommendedprimer concentration (Table 1(b))


Real-time RT-PCR assays were performed on the ther-mocycler Rotor Gene Q MDx and Mx3005P using the samethermocycling conditions for the five kits (ie 45 cycles of30 sec-94∘C 30 sec-55∘C and 30 sec-72∘C) For each testedkit the DNA polymerase activation step was performed asrecommended by the supplier (Table 1(c))

234 Data Collection and Analysis PCR efficiency and coef-ficient of determination (1198772) values were calculated by thesoftware Rotor Gene Q Series and Mx3005Pro respectively

Negative and positive controls were included in eachassay in which a threshold setting (Ct) of 003 was used as thereference for the Rotor Gene QMDx thermocycler A thresh-old setting of 300 was used as the reference for the Mx3005Pthermocycler for all tested kits except for one two-step kit(Express) for which a threshold setting of 005 was used

24 Determination of the Performance of the Method Theperformance of the method was determined for all testedkits following OIE recommendations [20] and those of theFrench Agency for Standard Operating Procedure for PCRexperimental design [21] Performance testingwas conductedfor the two real-time platforms

241 Determination of PCR Efficiency A 106 to 103 copies120583Ldilution range corresponds to the linear dynamic range forthe ten tested kits To determine the PCR efficiency foreach tested kit a standard curve was plotted based on Ctvalues obtained from samples containing 106 to 103 copies120583Lagainst the log of the estimated RNA copy number in thesample The coefficient of determination (1198772) was calculatedand considered as suitable when not lower than 099 Theslope of the standard curves was used for the determinationof PCR efficiency (119864 = 10[minus1slope] minus 1) The efficiency wasconsidered satisfactory when it is not lower than 90 and notabove 110 [22]

242 Determination of the Limit of Detection at 95 Thelimit of detection at 95 (LOD 95) which corresponds tothe lowest RNA concentration that could be detected with95 confidence was calculated using the inverse standardnormal distribution calculation [21] The LOD 95 wascalculated using the Probit analysis based on the results ofthree runs of eight repetitions of six dilutions each of EBLV-1RNA in RNase-free water (119899 = 144 samples tested) The sixdilutions (cDNA and RNA) were selected surrounding thelimit of detection of real-time PCR assays

On the Rotor Gene Q MDx gene cycler 103 to 25 cop-iessample were tested with the kit Rotor Gene-1 step 103to 50 copiessample for qScript-1 stepSSIII Platinium-1 step500 to 10 copiessample for Verso-1 step 50 to 1 copiessamplefor Quantitect-1 step 250 to 5 copiessample for Perfecta 250to 5 copiessample for QuantiTect 102 to 25 copiessamplefor Maxima 100 to 05 copiessample for Sso Fast and 100 to05 copiessample for Express

On Mx3005P cycler 2500 to 100 copiessample weretested with the kit qScript-1 step 250 to 5 copiessample forSSIII Platinium-1 stepVerso-1 step 50 to 1 copiessample forRotor Gene-1 stepQuantitect-1 step 500 to 10 copiessample

for QuantiTect 250 to 5 copies120583L for MaximaSso FastPer-fecta 250 to 5 copiessample for Perfecta and 100 to 25 cop-iessample for Express

Three independent assays were undertaken under identi-cal laboratory conditions

243 Evaluation of Reproducibility To evaluate the repro-ducibility of the ten kits inter- and intraspecific assay repro-ducibility were assessed using the coefficient of variation(CV) of theCt values for the lowest RNAconcentration show-ing 100 positive results The intra-assay repeatability wasdetermined using three independent runs of eight replicatesof EBLV-1 (119899 = 24 samples tested) The interassay repeata-bility was assessed for the same serial replicates in threeindependent assays using identical laboratory conditionsCVs expressed as percentages were obtained by dividing thestandard deviation of each tested sample by the mean of thecycle threshold values and multiplying that result by 100

3 Results

31 Differences in RNADetection Levels Using Two-StepAssays

311 Reverse Transcription Kits Of the five reverse transcrip-tion kits tested in combination with the SSoFast kit for cDNAamplification the SuperScript Vilo yielded greater sensitivityand resulted in a detection limit of 10 copies120583L (3 positivesamples out of 3 tested) The efficiency of amplification wasof 98The four other RT kits (ie kits Quantitect MaximaSS Vilo and qScript) resulted in a detection of 100 copies120583LThe amplification efficiency varied between 98 and 104The 119910-intercept for all reverse transcription kits tested incombination with the qPCR SsoFast kit varied between 3513and 3746

312 Two-Step PCR Kits All optimised two-step qPCRassays tested in combination with the Iscript RT showed sim-ilar sensitivity and enabled detection of at least 100 copies120583Lof EBLV-1 on the Rotor Gene Q MDx gene cycler Theefficiency of amplificationwhichwas satisfactory for all testedkits varied between 90 (ie kit Perfecta) and 99 (ie kitsSSo Fast Quantitect SG Maxima SG and Express) with an119910-intercept varying between 3603 and 3857

313 Comparison of Two-Step RT-qPCR Kits Provided bythe Same Manufacturer Table 2 details the results of thecomparison of the five two-step kits

(i) Rotor Gene Q MDx Of the five PCR kits tested incombination with the reverse transcriptases provided by thesame manufacturer one RT-qPCR assay (SuperScript Vilo incombination with qPCR Express kit) yielded greater sensitiv-ity and showed a detection of at least 10 copies120583L (Table 2)All amplicons investigated with the two-step method usingqPCR kits and RT kits from the same provider showedsatisfactory efficiency of amplification (100 plusmn 10) The 119910-intercept for all tested kits varied between 3473 and 3955

(ii) Mx3005P Of the five tested RT-qPCR kits one RT-qPCRassay (Maxima RTqPCRMaxima) yielded greater sensitivity


Table2Com

paris

onof

RT-qPC

RSybrGreen

kits(tw

o-ste

p)provided

bythes

amem

anufacturerRe

sults

offived

ifferentq

PCRkitson

arange

ofseventriplicated

ilutio

nsof

EBLV

-1RN

A

Testingof

different

RT-qPC

Rkits(tw

o-ste

p)provided

bythes

amem

anufacturerlowastlowastlowastlowast

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

IScriptR

T+qP

CRSsoF

ast

QuantiTectR

T+qP

CRQuantitect

Maxim

aRT+qP

CRMaxim

aSS

ViloRT

+qP

CREx

press

qScriptR

T+qP

CRPerfe

cta

AverageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowast

106

5010

51658plusmn013

077

1947plusmn007

034

1650plusmn015

092

1501plusmn

005

035

1609plusmn003

019

1896plusmn004

021

2279plusmn026

115

1747plusmn009

052

1749plusmn010

054

1799plusmn029

159

105

5010

41993plusmn009

045

2289plusmn010

042

1950plusmn006

033

1821plusmn

005

026

1979plusmn003

013

2217plusmn006

025

2594plusmn009

035

2116plusmn029

138

2095plusmn003

015

2127plusmn015

072

104

5010

32328plusmn005

020

2589plusmn006

025

2295plusmn028

123

2168plusmn017

076

2318plusmn016

067

2575plusmn005

018

2960plusmn042

143

2429plusmn029

039

2440plusmn012

049

2455plusmn008

033

103

5010

22661plusmn

021

080

2964plusmn032

107

2643plusmn026

098

2481plusmn

005

019

2694plusmn008

030

2916plusmn023

080

3210plusmn019

060

2778plusmn030

109

2803plusmn074

263

2784plusmn012

042

102

5010

13045plusmn013

042

3220plusmn041

126

3059plusmn045

146

2833plusmn064

225

3140plusmn053

168

3238plusmn061

189

3512plusmn010

028

3046plusmn092

303

3105plusmn038

123

3135plusmn070

222

101

5010

023lowastlowast

13lowastlowast

23lowastlowast

3152plusmn13

3420

13lowastlowast

13lowastlowast

13lowastlowast

3276plusmn070

212

13lowastlowast

23lowastlowast

105

13lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

23lowastlowast

Efficiency

99

99

100

101

90

96

107

97

93

102

1198772

0999

0997

0997

0999

0999

099

9099

1099

7099

4099

8

119910-in

tercept

3665

3955

3631

3473

3767

3938

4183

3801

3850

3801

lowastCV

()=

standard

deviationmeanCttimes100

CVwas

notcalculated

whensomeo

fthe

parallelsfailedto

amplify

lowastlowast119883119883

num

bero

fpositivesnu

mbero

fexp

erim

ents

lowastlowastlowastAv

erageC

tvaluesmeanplusmnstandard

deviation

lowastlowastlowastlowastAllPC

Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)


and showed a detection of at least 10 copies120583L All RT-qPCRassays showed satisfactory efficiency varying between 93 and107 with detection of at least 100 copies120583LThe 119910-interceptvaried between 3801 and 4183 for all tested kits

32 Differences in RNA Detection between the Five Kits TestedUsing the One-Step Method Table 3 details the results of thecomparison of the five one-step kits

(i) Rotor Gene Q MDx Three optimised qRT-PCR assaysrevealed poor sensitivity and enabled detection of at least103 copies120583L Of the five kits tested two kits (QuantiTect-1 step and Verso-1 step) yielded greater sensitivity with thedetection of at least 10 copies120583L (3 positive samples out of3 tested) All kits showed a satisfactory efficiency varyingbetween 94 and 104The119910-intercept varied between 3278and 3767 for the five tested one-step kits

A high presence of Primer-Dimer was shown for lowRNA concentrations for three kits SSIII Platinium-1 stepVerso-1 step and qScript-1 step

(ii) Mx3005P Of the five one-step kits tested using theMx3005P cycler three optimised one-step kits (ie Quanti-tect-1 step Rotor Gene-1 step and SSIII Platinium-1 step)revealed good sensitivity with the detection of at least10 copies120583L The two other optimised one-step assays (ieVerso-1 step and qScript-1 step) revealed poor sensitivitywith a detection of 102 and 103 copies120583L respectively Theamplification efficiencywas satisfactory for all kits (94ndash100)except for the qScript one-step kit (efficiency of 87) The 119910-intercept varied between 3680 and 4074 for all tested kitsexcept for the qScript kit (Intercept of 4074)

33 Comparison of Assay Performance

331 Limit of Detection at 95 for One-Step and Two-StepMethods The results of LOD 95 for the ten kits tested usingtwo-step and one-step methods are recorded in Table 4

(i) Rotor Gene Q MDx The results showed an LOD 95 fortwo-step and one-step kits ranging from 41 to 171 copies120583Land from 20 to 497 copies120583L respectively (Table 4) Ofthe five commercial master mixes tested using the two-stepqPCR the RT SuperScript Vilo in combination with theExpress kit yielded greater sensitivity with an LOD 95 of41 copies120583L Maxima in combination with the RT Maximaand Perfecta in combination with the RT qScript rankedsecond and third with detection of 78 and 88 copies120583Lrespectively followed by the SsoFast and QuantiTect kitswhich demonstrated the poorest levels of detection with anLOD 95 of 110 and 171 copies120583L respectively

In contrast the one-step qRT-PCR kit Quantitect-1 stepyielded the best levels of detection with an LOD 95 of20 copies120583L The four other tested one-step kits yieldedpoor detection with an LOD 95 varying between 127 and497 copies120583L

(ii) Mx3005P The LOD 95 for one-step and two-step kitsranged from 15 to 981 copies120583L and 45 to 160 copies120583L

respectivelyOf the five commercialmastermixes tested usingthe two-step qPCR the RT SuperScript Vilo in combinationwith the Express kit yielded greater sensitivity with an LOD95 of 45 copies120583L Perfecta in combination with qScriptRT and Maxima in combination with RT Maxima rankedsecond and third with a detection of 67 and 76 copies120583Lrespectively followed by the the SsoFast and QuantiTect kitswhich demonstrated the poorest levels of detection with anLOD 95 of 88 and 160 copies120583L respectively

Of the five tested one-step kits two kits (ie Rotorgene-1 step and Quantitect-1 step) yielded the best levelsof detection with an LOD 95 of 15 and 22 copies120583Lrespectively SSIII Platinium-1 step ranked third with an LOD95 of 50 copies120583L followed by Verso-1 step (LOD 95 of101 copies120583L) and qScript-1 step (981 copies120583L)

332 Reproducibility of One-Step and Two-Step MethodsTable 5 shows the analysis results of reproducibility of one-step and two-step assays

(i) Rotor Gene Q MDx For all optimised one-step or two-step PCR assays the interassay CVs were lower than 10demonstrating good reproducibility for all tested kits [23]The interassay CVs for the two-step kits and the one-step kitsranged from 174 to 295 and 135 to 427 respectively whilethe intra-assay CVs ranged from 135 to 35 and 079 to 672for two-step and one-step kits respectively (Tables 5(a) and5(b)) In analysing the intra-assay CVs a somewhat similarvariability was shown for two-step and one-step reactions

The interassay CVs for the most efficient two-step kit(ie SuperScript Vilo in combination with qPCR Express)and the most efficient one-step kit (ie Quantitect-1 step)ranged from 195 to 336 respectively while the intra-assayCVs ranged from 185 to 211 and 079 to 216 for two-step andone-step kits respectively (Tables 5(a) and 5(b))

(ii) Mx3005P For all optimised one-step or two-step PCRassays the interassay CVs were lower than 10 demonstrat-ing good reproducibility for all tested kitsThe interassay CVsfor the two-step and one-step kits ranged from 214 to 33and 084 to 45 respectively while the intra-assay CVs rangedfrom 123 to 422 and 038 to 47 for two-step and one-stepkits respectively

34 Practicability of the Different Assays Costs and TimeTable 6 shows a practicability assessment of the differentreal-time assays including a comparison of cost pipettingsteps and thermocycling times Total reaction times for theten real-time assays ranged from 2h42 to 3h08 for two-stepkits and 2h18 to 2h57 for one-step kits with the one-stepSSIII Platinium kit requiring the shortest time and two-stepMaxima kit the longest regardless the machine used Thekits were ranked from 1 to 10 based on the cost per PCRreaction in increasing order from C108 (least expensive =1) to C1225 (most expensive = 10) Overall of the 10 mastermix kits tested the one-step kits were cheaper than the two-step kits (Table 6) For the one-step kits qScript was theleast costly followed by the Rotor Gene-1 step Verso-1 stepSSIII Platinium-1 step and the Quantitect-1 step which was


Table3Com

paris

onof

qRT-PC

RSY

BRGreen

(one-step)

kits

Results

offived

ifferentq

RT-PCR

kitson

arange

ofseventriplicated

ilutio

nsof

EBLV

-1in

vitro

RNAtranscrip

tsTestingof

different

qRT-PC

Rkits(one-step)

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Quantitect-1ste

pRo

torG

ene-1step

Verso-1step

SSIIIP

latinium-1ste

pqScript-1

step

AverageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowast

106

2010

61666plusmn008

045

1715plusmn005

029

1699plusmn006

038

1452plusmn019

032

1347plusmn003

019

1779plusmn024

132

1588plusmn022

136

1834plusmn032

172

1622plusmn004

022

1848plusmn004

020

105

2010

52006plusmn010

052

2062plusmn004

017

2040plusmn011

052

1790plusmn021

118

1655plusmn003

019

2113plusmn045

212

1942plusmn020

104

2160plusmn015

071

1964plusmn041

211

2238plusmn015

068

104

2010

42358plusmn014

058

2391plusmn

008

033

2382plusmn020

083

2143plusmn012

057

1986plusmn004

018

2460plusmn020

083

2287plusmn016

069

2517plusmn017

069

2308plusmn027

116

2611plusmn010

038

103

2010

32670plusmn024

091

2745plusmn028

101

2693plusmn003

011

2490plusmn023

092

2313plusmn016

067

2795plusmn026

093

2634plusmn033

124

2863plusmn030

106

2614plusmn010

038

2954plusmn025

086

102

2010

23042plusmn008

026

23lowastlowast

3019plusmn060

198

03lowastlowast

03lowastlowast

3198plusmn017

053

2977plusmn039

132

3174plusmn079

250

2893plusmn008

028

3107plusmn037

118

101

2010

13324plusmn029

087

13lowastlowast

3327plusmn15

3460

03lowastlowast

03lowastlowast

3434plusmn10

3299

3284plusmn044

133

23lowastlowast

2991plusmn

010

033

03lowastlowast

12

03lowastlowast

13lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

23lowastlowast

03lowastlowast

03lowastlowast

Efficiency

98

96

100

94

104

97

94

95

100

87

1198772

0998

0999

0999

0998

0999

099

6099

8099

7099

6099

8

119910-in

tercept

3689

3767

3699

3529

3278

3815

3680

3893

3893

4074

lowastCV

()=

standard


CVwas

notcalculated

whensomeo

fthe

parallelsfailedto

amplify

lowastlowast119883119883num

bero

fpositivesnu

mbero

fexp

erim

ents


erageC


deviation


Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)


Table4Detectio

nlim

itof

PCR(LOD95)for

two-ste

pandon

e-ste

pSY

BRGreen

RT-PCR

Two-ste

pRT

-qPC

RIScriptR

T+qP

CRSsoF

ast

BioR

adQuantiTectR

T+qP

CRQuantitect

QIAGEN

Maxim

aRT+qP

CRMaxim

aFE

RMEN

TAS

SSViloRT

+qP

CREx

press

INVITRO

GEN

qScriptR

T+qP

CRPerfe

cta

QUA

NTA

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

11055

171

8678

3941

2188

4488

44160

8076

3845

2267

34One-stepqR

T-PC

RQuantitect-1ste

pQIAGEN

RotorG

ene-1step

QIAGEN

Verso-1step

FERM

ENTA

SSSIIIP

latinium-1ste

pIN

VITRO

GEN

qScript-1

step

QUA

NTA

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

2040

497

994

127

254

321

641

406

812

2244

1530

101

202

50100

981

1962

lowastAllLO

D95results

werec

arrie

dou

tusin

gProb

itmod

ellowastAllPC

Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)


Table5Analysis

ofreprod

ucibilityof

one-ste

pandtwo-ste

pRT

-PCR

(a)

show

sfor

each

two-ste

pkittheintra-a

ndinterassay

reprod

ucibilityforsixdilutio

nsof

EBLV

-1teste

deighttim

esin

each

ofthreeind

ependent

runs

(119899=144samples)a

nd(b)sho

wsthe

inter-andintra-assayreprod

ucibilityfora

llteste

don

e-ste

pkits

(a)2-ste

pmetho

ds

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria

bilitylowastlowastlowast

Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CV ()lowastlowast

AverageC

tplusmnSD

CtCV

()lowastlowast

IScriptR

T+qP

CRSsoF

ast

100

3046plusmn041

135

83008plusmn057

190

82982plusmn041

138

83012plusmn046

154

3012plusmn052

174

503111plusmn093

299

63184plusmn10

1319

63080plusmn046

151

83125plusmn080

256

3120plusmn088

283

253249plusmn059

183

63160plusmn067

212

83127plusmn068

219

63179plusmn065

204

3177plusmn080

251

103238plusmn091

281

63269plusmn119

363

63197plusmn076

239

53235plusmn095

294

3237plusmn097

299

53314

mdash1

mdashmdash

03198plusmn005

015

33256plusmn005

015

3227plusmn058

181

05

3217

mdash1

mdashmdash

03211plusmn076

236

33214plusmn076

236

3213plusmn062

193

250

3071plusmn

049

161

830

70plusmn039

128

830

61plusmn

033

108

830

67plusmn041

132

3067plusmn040

129

100

3230plusmn074

228

83203plusmn090

281

83219plusmn040

124

83217plusmn068

211

3217plusmn069

214

5034

02plusmn093

273

63324plusmn048

144

83316plusmn093

287

83347plusmn079

235

3342plusmn085

255

2534

63plusmn10

2295

83378plusmn075

223

634

24plusmn088

258

634

21plusmn

089

259

3425plusmn093

272

103595plusmn11

2312

53526plusmn047

134

534

68plusmn00

63

180

63530plusmn074

209

3526plusmn090

256

53384plusmn062

184

3mdash

mdash0

mdashmdash

034

83plusmn060

171

3384plusmn062

184

QuantiTectR

T+qP

CRQuantitect

250

3137plusmn069

220

83145plusmn052

165

83122plusmn046

147

83135plusmn056

177

3135plusmn055

175

100

3246plusmn080

246

73272plusmn092

281

73390plusmn090

265

73303plusmn087

264

3303plusmn10

5318

503359plusmn060

179

43434plusmn232

675

53366plusmn079

235

63386plusmn12

4363

3387plusmn14

0413

253298plusmn074

224

43358plusmn118

351

53412plusmn207

608

43356plusmn13

3394

3356plusmn13

8410

103354plusmn018

054

33386plusmn061

180

23473plusmn062

179

23404plusmn047

138

3397plusmn065

192

5mdash

mdash0

3552

mdash1

mdashmdash

03552

mdash3552

mdash50

034

09plusmn036

135

836

37plusmn118

326

63297plusmn038

116

834

30plusmn15

419

234

30plusmn15

4448

250

3642plusmn046

643

83366plusmn080

239

83361plusmn

083

246

834

56plusmn19

7376

3456plusmn19

7569

100

3439plusmn234

257

834

60plusmn098

284

834

50plusmn091

265

834

50plusmn089

269

3450plusmn089

258

5036

28plusmn088

475

63507plusmn091

259

734

92plusmn10

4297

73538plusmn13

2344

3538plusmn13

2374

253597plusmn17

2230

536

28plusmn088

242

436

44plusmn078

214

536

23plusmn079

229

3623plusmn079

217

10mdash

mdash0

mdashmdash

03765plusmn079

210

23765plusmn079

210

3765plusmn079

210


(a)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CV ()lowastlowast

AverageC

tplusmnSD

CtCV

()lowastlowast

Maxim

aRT

+qP

CRMaxim

a

100

3183plusmn084

263

3138plusmn022

070

3009plusmn041

137

311plusmn049

157

311plusmn092

295

503335plusmn10

9326

83342plusmn088

264

53112plusmn064

205

83263plusmn087

265

3251plusmn

140

432

253367plusmn088

261

53277plusmn112

340

63247plusmn072

221

73297plusmn090

274

3290plusmn099

302

103716plusmn464

1249

33400plusmn14

1416

43435plusmn13

3388

53517plusmn246

684

3494plusmn263

754

53544plusmn024

068

23865

mdash1

3458plusmn12

4357

33622plusmn074

212

3555plusmn17

6496

25

mdashmdash

03314

mdash1

3347plusmn17

6526

23330plusmn17

6526

3336plusmn12

6377

250

2928plusmn058

199

829

05plusmn038

130

829

04plusmn039

135

829

12plusmn045

155

2912plusmn045

156

100

3053plusmn056

185

830

17plusmn048

159

830

31plusmn

043

143

830

34plusmn050

162

3034plusmn050

164

503154plusmn12

5397

83188plusmn13

7429

83172plusmn056

177

83171plusmn

108

334

3171plusmn

108

340

253300plusmn115

348

63238plusmn079

243

73238plusmn082

254

73256plusmn092

282

3256plusmn092

281

103308plusmn064

193

33302plusmn13

8417

43340plusmn14

6438

43317plusmn115

349

3317plusmn11

5347

503303plusmn015

045

23355

mdash1

3299

mdash1

3315plusmn028

045

3315plusmn028

085

qScriptR

T+qP

CRPerfe

cta

250

2704plusmn024

088

82675plusmn026

098

82677plusmn024

090

82685plusmn025

092

2685plusmn027

101

100

3054plusmn054

176

82984plusmn054

182

83046plusmn10

7350

83028plusmn072

236

3028plusmn079

261

503203plusmn290

905

73072plusmn089

292

83078plusmn082

267

83118plusmn15

4488

3114plusmn17

7568

253250plusmn098

302

73241plusmn

133

410

73363plusmn216

642

53285plusmn14

9452

3276plusmn14

9456

103222plusmn081

252

53287plusmn046

139

33270plusmn056

171

23260plusmn061

187

3251plusmn

069

211

53278plusmn015

045

2mdash

mdash0

3356plusmn15

5461

23318plusmn085

253

3318plusmn10

0303

250

2947plusmn022

074

829

49plusmn054

183

729

82plusmn038

129

829

60plusmn041

129

2960plusmn041

139

100

3099plusmn10

9352

83114plusmn12

6404

83102plusmn052

169

83105plusmn096

308

3105plusmn096

311

503187plusmn060

188

83137plusmn057

180

83313plusmn11

2337

83212plusmn10

8235

3212plusmn10

8335

253335plusmn299

896

73249plusmn068

211

83271plusmn

087

265

83283plusmn17

2457

3283plusmn17

2524

103300plusmn049

147

53345plusmn018

053

23298plusmn074

225

43307plusmn054

142

3307plusmn054

165

53304plusmn10

1306

23303plusmn074

223

23359

mdash1

3314plusmn067

265

3314plusmn067

203


(a)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CV ()lowastlowast

AverageC

tplusmnSD

CtCV

()lowastlowast

SSViloRT

+qP

CREx

press

100

2768plusmn059

213

82782plusmn055

197

82759plusmn029

106

82770plusmn048

172

2770plusmn048

174

502942plusmn062

211

82927plusmn054

185

82921plusmn

060

206

82930plusmn059

201

2930plusmn057

195

253038plusmn067

221

63022plusmn12

4411

82960plusmn048

162

83007plusmn080

264

3004plusmn090

300

103136plusmn049

158

73130plusmn071

227

73192plusmn078

245

63153plusmn066

210

3151plusmn

069

219

53149plusmn053

168

43163plusmn098

308

43164

mdash1

3159plusmn075

238

3157plusmn068

216

05

mdashmdash

0mdash

mdash0

3159

mdash1

3159

mdash3159

mdash100

3178plusmn053

167

830

13plusmn032

107

830

75plusmn065

211

830

89plusmn085

162

3089plusmn085

276

503296plusmn042

129

83134plusmn050

161

83149plusmn097

307

83193plusmn099

199

3193plusmn099

309

253372plusmn10

5310

73215plusmn098

306

83209plusmn022

069

63266plusmn113

228

3266plusmn11

3346

1034

44plusmn060

174

73361plusmn

046

135

53330plusmn043

129

63383plusmn071

146

3383plusmn071

209

53521plusmn

053

151

73264plusmn036

111

53299plusmn006

017

23397plusmn13

5093

3397plusmn13

5398

25

3956plusmn18

6471

334

35

13355plusmn057

169

336

24plusmn332

320

3624plusmn332

916

Theintra-andinterassay

varia

bilitya

ndcoeffi

ciento

fvariatio

nswerec

alculated

bytheformulasinter-assay

CV=sta

ndarddeviationofallreplicates(meanCt-value

ofallreplicates)times

100intra-assayC

V=mean

oftheC

Vso

fallruns

lowastAv

erageC


deviation

lowastlowastCV

()=

standard


lowastlowastlowastAllPC

Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)

(b)1stepmetho

ds

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CVlowastlowast

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Quantitect1step

502976plusmn064

216

82894plusmn055

189

82927plusmn023

079

82932plusmn047

161

2932plusmn059

106

253090plusmn088

286

82984plusmn10

9365

83098plusmn076

245

83057plusmn091

299

3057plusmn10

3202

103172plusmn10

4326

73205plusmn10

6332

73230plusmn061

188

83203plusmn090

282

3204plusmn090

336

53334plusmn12

0360

63238plusmn074

228

73285plusmn084

255

73286plusmn092

281

3283plusmn096

281

25

3251plusmn

060

185

43390plusmn033

096

43445plusmn15

2442

53362plusmn082

241

3368plusmn12

7377

13338plusmn096

288

43203plusmn026

080

33292plusmn047

143

53277plusmn056

170

3285plusmn079

242

5030

92plusmn047

152

83123plusmn12

9040

83111plusmn13

2041

83108plusmn13

8043

3108plusmn043

138

253177plusmn073

229

83192plusmn14

9048

83191plusmn

093

030

83187plusmn15

7050

3187plusmn051

161

103260plusmn047

144

83281plusmn

061

187

53249plusmn065

199

83263plusmn058

177

3263plusmn057

175

53369plusmn085

253

73382plusmn291

098

53328plusmn250

083

63360plusmn265

089

3357plusmn086

257

25

3436plusmn073

214

53365plusmn18

8063

23327plusmn18

8062

33376plusmn19

6066

3380plusmn080

236

134

55plusmn17

7513

334

97plusmn117

041

53392plusmn341

116

134

48plusmn324

111

3445plusmn13

7397


(b)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CVlowastlowast

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

RotorG

ene1

step

1000

2640plusmn045

169

82374plusmn040

167

82701plusmn

118

438

82572plusmn16

2258

2572plusmn16

2632

500

2749plusmn046

166

82429plusmn034

139

82742plusmn10

4378

82640plusmn16

6228

2640plusmn16

6628

250

2873plusmn076

265

82490plusmn049

196

82927plusmn060

204

72756plusmn209

222

2756plusmn209

758

100

3070plusmn021

067

32550

mdash1

3119plusmn066

211

23000plusmn224

139

3000plusmn224

746

503031

mdash1

mdashmdash

0mdash

mdash0

3031

mdash3031

mdash25

3112plusmn050

161

2mdash

mdash0

mdashmdash

03212plusmn050

161

3212plusmn050

161

5028

87plusmn099

341

829

25plusmn074

252

82797plusmn065

233

828

70plusmn079

276

2870plusmn094

329

2529

49plusmn10

1344

830

36plusmn070

229

828

57plusmn039

136

829

47plusmn070

236

2947plusmn10

3350

1030

29plusmn12

0398

830

61plusmn

144

470

828

76plusmn040

139

829

89plusmn10

1336

2989plusmn13

4450

530

82plusmn10

6343

63147plusmn13

0412

629

90plusmn040

135

830

73plusmn092

297

3065plusmn11

2366

25

3087plusmn114

368

63169plusmn057

181

529

10plusmn037

126

530

55plusmn069

225

3057plusmn13

1429

13122

mdash1

3153

mdash1

3201plusmn

044

137

53159plusmn044

137

3183plusmn048

151

Verso1step

500

2650plusmn018

068

82656plusmn014

053

82679plusmn023

085

82661plusmn

018

069

2661plusmn

022

082

250

2743plusmn018

065

82796plusmn027

098

82789plusmn036

127

82776plusmn027

097

2776plusmn036

130

100

2894plusmn050

172

82942plusmn053

180

82991plusmn

118

396

82943plusmn074

249

2943plusmn087

295

503049plusmn044

143

73138plusmn086

274

83129plusmn15

2487

83105plusmn094

301

3108plusmn10

9350

253282plusmn114

346

73290plusmn052

157

53250plusmn092

282

73274plusmn086

262

3272plusmn090

274

103342plusmn097

291

33522plusmn10

5299

23442

mdash1

3435plusmn10

1295

3419plusmn118

344

250

2939plusmn048

164

829

40plusmn023

079

829

10plusmn053

183

829

20plusmn042

142

2930plusmn044

150

100

3055plusmn025

080

830

32plusmn038

125

830

02plusmn024

082

630

29plusmn029

096

3032plusmn036

119

503116plusmn050

159

83110plusmn052

168

830

87plusmn042

135

73104plusmn048

154

3105plusmn048

153

253171plusmn

032

102

73161plusmn

042

134

83135plusmn058

184

83156plusmn044

140

3155plusmn046

147

103186plusmn054

169

73176plusmn029

091

73165plusmn064

201

43176plusmn049

154

3177plusmn046

145

53309plusmn098

297

53313

13343plusmn074

223

43322plusmn086

260

3326plusmn080

239


(b)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CVlowastlowast

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

qScript1

step

1000

2324plusmn012

053

82297plusmn024

103

82321plusmn

009

037

82314plusmn015

064

2314plusmn020

086

750

2364plusmn021

088

82329plusmn008

035

82352plusmn028

121

82348plusmn019

081

2348plusmn025

106

500

2446plusmn032

132

82406plusmn024

098

82407plusmn029

122

82419plusmn028

117

2419plusmn033

137

250

2538plusmn027

107

82493plusmn026

105

82514plusmn034

137

82515plusmn029

116

2515plusmn034

135

100

2626plusmn031

116

82579plusmn024

093

72595plusmn047

180

52600plusmn034

130

2602plusmn038

146

50mdash

mdash0

mdashmdash

02645plusmn018

070

22645plusmn018

070

2645plusmn018

070

2500

2813plusmn083

294

82778plusmn045

160

826

32plusmn030

113

82741plusmn

052

189

2741plusmn

097

353

1000

2878plusmn009

033

829

40plusmn15

8539

824

89plusmn030

122

82769plusmn066

231

2769plusmn223

805

750

2908plusmn024

082

828

92plusmn025

085

828

75plusmn011

038

828

92plusmn020

068

2892plusmn024

084

500

3069plusmn048

157

729

76plusmn055

186

82763plusmn056

201

529

36plusmn053

181

2966plusmn12

6424

250

3122plusmn023

073

730

05plusmn037

124

8mdash

mdash0

3064plusmn030

099

3060plusmn067

220

100

mdashmdash

030

00

mdash1

mdashmdash

030

00

mdash30

00

mdash

SSIIIP

latinium

1step

1000

2479plusmn024

097

82381plusmn

021

089

82446plusmn015

060

82435plusmn020

082

2435plusmn046

188

750

2518plusmn024

095

82430plusmn012

051

82497plusmn010

042

82482plusmn016

063

2482plusmn041

167

500

2666plusmn082

307

82536plusmn023

092

82619plusmn024

092

82607plusmn043

164

2607plusmn073

282

250

2866plusmn086

301

82838plusmn19

1672

82897plusmn059

205

82867plusmn112

393

2867plusmn12

2427

100

2917plusmn053

180

82778plusmn049

175

82816plusmn053

189

72837plusmn052

182

2838plusmn078

276

503095plusmn043

139

22911plusmn067

231

42993plusmn111

370

33000plusmn074

247

2979plusmn10

3347

250

2627plusmn026

099

826

70plusmn027

102

826

67plusmn042

156

826

55plusmn037

119

2655plusmn037

139

100

2722plusmn068

248

828

02plusmn039

138

82774plusmn034

121

82766plusmn058

169

2766plusmn058

209

502756plusmn053

194

828

26plusmn041

147

828

33plusmn040

141

828

05plusmn056

161

2805plusmn056

200

252787plusmn069

247

829

34plusmn055

189

828

58plusmn041

145

828

59plusmn082

194

2859plusmn082

285

1028

75plusmn078

270

629

44plusmn055

186

529

08plusmn033

114

729

07plusmn060

190

2907plusmn060

207

528

82plusmn015

053

428

71plusmn

021

072

328

88plusmn032

112

428

81plusmn

023

079

2881plusmn

023

079

Theintra-a

ndinterassay

varia

bilityandcoeffi

ciento

fvariatio

nswerec

alculated

bytheformulasinterassayCV

=standard

deviationof

allreplicates(meanCt-value


100intra-assayCV

=mean

oftheC

Vso

fallruns

lowastAv

erageC


deviation

lowastlowastCV

()=

standard



Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)


Table 6 Comparison of the 10 commercial master mixes withregard to the practicability of the different assays and the coststimes and number of pipetting steps

Ranking forcostPCRreactionlowast

Run duration(minutes) Number

ofpipettingsteps lowastlowast

RotorGene QMDx

Mx3005P

Two-step RT-qPCRSsoFast(Iscript) 9 242 253 12QuantiTect(QuantiTect) 8 254 300 14

Maxima (Maxima) 7 257 308 14Perfecta (qScript) 6 248 256 11Express (SS Vilo) 10 250 301 10

One-step qRT-PCRQuantitect-1 step 5 248 257 7Rotor Gene-1 step 2 239 248 7Verso-1 step 3 246 257 8SSIIIPlatinium-1 step 4 218 228 7

qScript-1 step 1 238 248 7lowastThe cost was calculated by dividing the cost for each commercial kit basedon the manufacturerrsquos list price in summer of 2013 by the number of PCRreactions that could be performed with each kit The kits were ranked from1 to 10 based on their cost per reaction with 1 corresponding to the leastexpensive and 10 to the most expensivelowastlowastPipetting steps include those corresponding to the preparation of themaster mix and the reverse transcription and PCR steps

the most expensive of all the tested kits The Express mastermix kit with SuperScript Vilo was the most expensive perassay of the two-step kits evaluated in this study Of thetwo-step kits the least expensive was Perfecta followed byMaxima QuantiTect and Sso Fast

4 Discussion

The first practical real-time PCR the 51015840 nuclease assay wasestablished in 1993 combining the exponential PCR amplifi-cation of a specific transcript with the monitoring of newlysynthesised DNA in each PCR cycle performed [24] Overthe past several years real-time PCR has become the leadingtool for the detection and quantification of nucleic acidsSince the first publications of real-time RT-PCR describingthe rapid detection of rabies virus in 2004 [10 25] variousprotocols for molecular diagnosis of Lyssavirus using SYBRGreen andor TaqMan probes have been published [11 1216 26 27] Although molecular techniques are not currentlyrecommended by the international authorities for routinediagnosis of rabies real-time PCR is increasingly used Theavailability of commercial master mix kits has increasedover the past decade and diagnostic laboratories willing toimplement real-time PCR are now faced with identifying themost suitable kit for their real-time PCR applications It hasbeen demonstrated that the choice ofmastermix reagents the

concentration of reaction mix components such as primersprobes analytical platforms linked to the real-time detectionplatform the different chemistries and the methodologyused (one-step versus two-step systems) can impact the sensi-tivity and consistency of results for real-time PCR assays [28]

The objective of this study was to evaluate the sensitiv-ity efficiency and reproducibility of one-step and two-stepsystems for the detection of EBLV-1 This virus species wasselected for this study for the generation of in vitro RNAtranscript This bat virus is widely isolated on Europeanbats the vast majority of rabies cases in bats in Europebeing attributed to EBLV-1 The increased interest in batsas a potential host for zoonotic viruses has resulted in thedetection of many new Lyssavirus species in the past ten years[29] In our study we selected five different cDNA generationsystems and ten commercial kits that is five one-step kits andfive two-step kits These kits are commonly used by NationalReference Laboratories that conduct rabies diagnosis (datanot shown) Our results demonstrated that there was littledifference in reaction efficiency1198772 values or intra- and inter-reproducibility between one-step and two-step methods Inour experience regardless of the real-time cycler used one-step qRT-PCR showed better limit of detection at 95 thanthe optimised two-step assays using pan Lyssavirus primers

The RT reaction is a key step in quantitative two-stepRT-qPCR RT priming strategy dynamic range and the RTenzyme which are prime considerations for a successfulreal-time RT-PCR assay are all important for ensuring thatmRNA expression levels are accurately represented in theresulting cDNA [30] For many quantitative applicationsthe MMLV RT (Moloney Murine Leukemia Virus reversetranscriptase) is described as the enzyme of choice as itscDNA synthesis rate is up to 40-fold greater than that ofAMV(AvianMyeloblastosisVirus) reverse transcriptase [23]Few results comparing the sensitivity of reverse transcriptasesare currently available [31] In this study five different cDNAgeneration systems were compared (eg 4 different MMLVRTs and a mix of Omniscript and Sensiscript RTs) eachkit containing unspecific target gene primers (eg randomhexamer (119899 = 3 kits) a mix of random hexamers andoligo dT (119899 = 2 kits)) By varying the RT chemistry andthe RT priming strategy we showed that the SuperScriptVilo reverse transcriptase which is an engineered version ofMMLV RT has provided the highest sensitivity with a gainof 1 log over the other systems of cDNA generation Theseresults are concordant with those of the literature [23 32 33]demonstrating the key role of the reverse transcriptase kit ina sensitive real-time two-step assay and providing the highestMMLV reverse transcriptase performance

In addition to assay performance criteria another goalof this study was to compare the performance of each kitwith the cost the practicability of each tested master mixand the presence or absence of Primer-Dimer for low DNAconcentrations Interestingly the one-step QuantiTect SYBRGreen RT-PCR kit which was shown the more efficient one-step kits is also one of the most expensive and required thelongest thermocycling time while the combination of Super-Script VILO reverse transcriptase and the qPCR Express kitwhich was found to be the most efficient of the two-step kits


using the two thermocyclers is also the most expensive ofthe two-step kits No difference in run duration was shownbetween the best two-step kits SuperScript VILOqPCRExpress and Maxima H Minus RTqPCR Maxima and theone-step kit QuantiTect SYBR Green RT-PCR confirmingthat all the kits are suitable for RNA detection regardlessof the thermocycling times However when using the ther-mocycler Rotor Gene MQDx the real-time one-step SYBRGreen assays with universal pan Lyssavirus revealed a highpresence of Primer-Dimers for three out of five one-step kitstested especially when RNA concentration was low (below100 copies120583L) compared to two-step kits (data not shown)The results of such techniques should be interpreted withattention since interpretation can be difficult especially forlow concentrations of samples

Few studies have been performed comparing one-stepand two-step methods the chemistries (Taqman probes ver-sus SYBR green) or the platforms for detection of infectiousagents There are advantages and disadvantages to bothmethods as well as to the use of SYBR green or fluorogenicprobes The main advantage of the one-step method is thatit is less time consuming and requires no user interventiontherefore minimising the chance of pipetting errors and crosscontamination The main advantage to two-step RT-qPCR isthat typically random hexamer or oligo dT primers are usedin an RT reaction in a separate tube allowing the archiving ofsamples for further testing of different species of rabies or forfuture testing of other genesMoreover in theory the one-stepmethod should offer higher or at least the same sensitivityas a two-step method because of the use of gene-specificprimers In our experience of the five one-step method kitstested we found three that yielded satisfactory analyticalsensitivity with an LOD 95 of 15ndash50 copies120583L Our resultsare concordant with previous results undertaken by Fischeret al [32] Based on the analysis of Ct values a study of real-time RT-PCRs on infected brain samples [32] showed thatthree one-step SYBR Green kits were more sensitive than thetwo two-step kits

The sensitivity of real-time PCR assays is mainly deter-mined by the master mix reagents but also by other fac-tors such as the platform used The performance of thethermocycler has a critical influence on PCR efficiencyPrevious studies have reported significant variability in PCRefficiency and results among different types of thermocyclers[34ndash36] Considering the various types of real-time equip-ment used in National Reference Laboratories involved inrabies diagnosis the ten master mixes were evaluated forintermediate reproducibility on two real-time platforms withvarying throughput capacities These two platforms differon thermal engine (heating block technology based on thePeltier effect versus heat exchange technology which permitmore rapid thermal ramp rates than blocks) Both real-time platforms are currently used by half of the NationalReference Rabies laboratories (ie 919) We showed thatthe more efficient one-step kit was the one-step Quanti-Tect SYBR Green RT-PCR kit with an LOD 95 of 20and 22 copies120583L on the two real-time cyclers ldquoRotor GeneQMDx Mx3005Prdquo respectively The reverse transcriptionkit SuperScript VILO in combination with the EXPRESS

SYBR GreenER kit is revealed to be the most sensitive ofthe two-step kits offering a limit of detection of 41 and45 copies120583L respectively Previous studies have reported theinfluence of the thermocyclers Buzard et al demonstratedthat certain combinations of commonly used master mixesand instruments are not as reliable as others for the detectionof lowDNA concentrations [28] In our experiment the LOD95 of Rotor Gene-1 step from QIAGEN operated with theRotor Gene Q MDX thermocycler was 497 copiessamplewhereas it resulted in 15 copiessample with the Mx3005Pthermocycler Our results demonstrated the pivotal influenceof the thermocycler on PCR performance and that a mastermix that showed high performance for one machine may notnecessarily work on another Our findings further show thatPCR protocols need to be optimised for different instrumentsand that validation of PCR protocols should include theintermediate reproducibility studies as recommended by theWorld Organization for Animal Health [37]

PCRprotocols need to be validated since a large variety ofparameters can influence the outcome of PCR amplificationPCR results generated by one lab are hardly reproducibleby another even under identical assay conditions Due tothe high sensitivity of real-time PCR assays if they are notperformed by highly trained molecular biology staff there isa high risk of sample or test contamination resulting in falsepositives For these reasons although these molecular testshave the highest level of sensitivity their use is currently notrecommended for routine postmortemdiagnosis of rabies [4]due to the high levels of false positive or false negative results[7] The considerable development of molecular techniquesin the 2000s has resulted in a wide variation in methodsand these techniques have become increasingly importantfor decision-making in rabies case management Howeverit is essential that laboratories employing PCR-based assaysapply very stringent laboratory quality control standards andthat they validate these highly sensitive techniques followinginternational guideline recommendations [37]

5 Conclusion

With the optimisation of PCR on two real-time instrumentsas well as the optimization of the RT step when testing thetwo-step method we showed that one-step PCR is moresensitive than the two-stepmethod regardless of themachineused We showed that the more efficient one-step kit wasthe one-step QuantiTect SYBR Green RT-PCR kit with anLOD 95 of 20 and 22 copies120583L on the two real-time cyclersldquoRotor Gene Q MDx Mx3005Prdquo respectively PCR assaysdemonstrated the crucial influence of the thermocycler onPCR performance for the detection of EBLV-1 despite theoptimisation of PCR assays and that a master mix whichperforms well on one machine may not necessarily work aswell on another In conclusion PCR protocols should be sys-tematically optimised for different instruments and validatedfollowing the recommendations of the World Organizationfor Animal Health


Abbreviations

Ct Threshold cycleCV Coefficient of variationEBLV-1 European bat Lyssavirus type 1LOD 95 Limit of detection at 95PCR Polymerase chain reactionRT Reverse transcription1198772 Coefficient of determination

Conflict of Interests

The authors declare that they have no competing interests

Acknowledgments

The authors wish to thank the European Commission andANSES for their financial support of this study The authorsalso wish to thankMelanie Biarnais andMarie-Jose Duchenefor their contributions

References

[1] ICTV ldquoInternational Committee on Taxonomy of Virusesrdquoin Vertebrate 2014 httpwwwictvonlineorgvirusTaxonomyasp

[2] H Badrane and N Tordo ldquoHost switching in Lyssavirus historyfrom the chiroptera to the carnivora ordersrdquo Journal of Virologyvol 75 no 17 pp 8096ndash8104 2001

[3] Anonyme Data Base QueriesmdashRabies Surveillancemdash1977ndash2013 WHO Collaboration Centre for Rabies Surveillance andResearch 2013

[4] World Organization for Animal Health ldquoRabiesrdquo in OIE Man-ual of Diagnostic Tests and Vaccines for Terrestrial Animals(Mammals Birds and Bees) 6th edition 2013

[5] A Sails ldquoApplications in clinical microbiologyrdquo in Real-TimePCR Current Technology andApplications J Logan K Edwardsand and N Sanders Eds chapter 13 pp 177ndash219 CaisterAcademic Press Norfolk UK 2009

[6] A R Fooks N Johnson C M Freuling et al ldquoEmergingtechnologies for the detection of rabies virus challenges andhopes in the 21st centuryrdquo PLoS Neglected Tropical Diseases vol3 no 9 article E530 2009

[7] E Robardet E Picard-Meyer S Andrieu A Servat and FCliquet ldquoInternational interlaboratory trials on rabies diagno-sis an overview of results and variation in reference diagnosistechniques (fluorescent antibody test rabies tissue cultureinfection test mouse inoculation test) and molecular biologytechniquesrdquo Journal of Virological Methods vol 177 no 1 pp15ndash25 2011

[8] E Picard-Meyer V Bruyere J Barrat E TissotM J Barrat andF Cliquet ldquoDevelopment of a hemi-nested RT-PCR methodfor the specific determination of European Bat Lyssavirus 1comparisonwith other rabies diagnosticmethodsrdquoVaccine vol22 no 15-16 pp 1921ndash1929 2004

[9] S A Nadin-DavisM Sheen andA IWandeler ldquoDevelopmentof real-time reverse transcriptase polymerase chain reactionmethods for human rabies diagnosisrdquo Journal of Medical Virol-ogy vol 81 no 8 pp 1484ndash1497 2009

[10] G J Hughes J S Smith C A Hanlon and C E RupprechtldquoEvaluation of a TaqMan PCR assay to detect rabies virus RNA

influence of sequence variation and application to quantifica-tion of viral loadsrdquo Journal of Clinical Microbiology vol 42 no1 pp 299ndash306 2004

[11] J Coertse J Weyer L H Nel and W Markotter ldquoImprovedPCR methods for detection of african rabies and rabies-relatedlyssavirusesrdquo Journal of Clinical Microbiology vol 48 no 11 pp3949ndash3955 2010

[12] D T S Hayman A C Banyard P RWakeley et al ldquoA universalreal-time assay for the detection of Lyssavirusesrdquo Journal ofVirological Methods vol 177 no 1 pp 87ndash93 2011

[13] P R Wakeley N Johnson L M McElhinney D MarstonJ Sawyer and A R Fooks ldquoDevelopment of a real-timeTaqMan reverse transcription-PCR assay for detection anddifferentiation of lyssavirus genotypes 1 5 and 6rdquo Journal ofClinical Microbiology vol 43 no 6 pp 2786ndash2792 2005

[14] S Wacharapluesadee P Phumesin P Supavonwong P Khaw-plod N Intarut and T Hemachudha ldquoComparative detectionof rabies RNA by NASBA real-time PCR and conventionalPCRrdquo Journal of Virological Methods vol 175 no 2 pp 278ndash282 2011

[15] T Nagaraj J P Vasanth A Desai A Kamat S N Madhusu-dana and V Ravi ldquoAnte mortem diagnosis of human rabiesusing saliva samples comparison of real time and conventionalRT-PCR techniquesrdquo Journal of Clinical Virology vol 36 no 1pp 17ndash23 2006

[16] A G Szanto S A Nadin-Davis R C Rosatte and B NWhiteldquoRe-assessment of direct fluorescent antibody negative braintissues with a real-time PCR assay to detect the presence ofraccoon rabies virus RNArdquo Journal of Virological Methods vol174 no 1-2 pp 110ndash116 2011

[17] A R Fooks L M McElhinney D Horton et al ldquoMoleculartools for rabies diagnosis in animalsrdquo in Compendium of theOIE Global Conference on Rabies Control Incheon-Seoul KoreaSeptember 2011 pp 75ndash85 OIE Paris France 2012



[20] OIE ldquoPrinciples and methods of validation of diagnostic assaysfor infectious diseasesrdquo inManual of Diagnostic TestsampVaccinesfor Terrestrial Animals p 16 World Organisation for AnimalHealth Paris France 2013

[21] AFNOR ldquoMethodes drsquoanalyses en sante animale PCR (Reactionde polymerisation en chaıne)mdashpartie 2 exigences et recom-mandations pour le developpement et la validation de la PCRen sante animale XP U 47-600-2rdquo Normes amp Recueils 2011

[22] M Pfaffl J Vandesompele and M Kubista ldquoData analysis soft-warerdquo in Real-Time PCR Current Technology and ApplicationsJ Logan K Edwards and and N Saunders Eds chapter 5 pp65ndash83 Caister Academic Press Norfolk UK 2009

[23] M Pfaffl ldquoQuantification strategies in real-time PCRrdquo in AndashZ of Quantitative PCR S Bustin Ed chapter 3 pp 87ndash112International University Line La Jolla Calif USA 2004

[24] R Higuchi C Fockler G Dollinger and R Watson ldquoKineticPCR analysis real-time monitoring of DNA amplificationreactionsrdquo Nature Biotechnology vol 11 no 9 pp 1026ndash10301993


[25] V Shankar R A Bowen A D Davis C E Rupprecht and T JOrsquoShea ldquoRabies in a captive colony of big brown bats (Eptesicusfuscus)rdquo Journal of Wildlife Diseases vol 40 no 3 pp 403ndash4132004

[26] B Hoffmann C M Freuling P R Wakeley et al ldquoImprovedsafety for molecular diagnosis of classical rabies viruses by useof a TaqMan real-time reverse transcription-PCR lsquodouble checkrsquostrategyrdquo Journal of Clinical Microbiology vol 48 no 11 pp3970ndash3978 2010

[27] M Fischer C M Freuling T Muller et al ldquoMolecular double-check strategy for the identification and characterization ofEuropean Lyssavirusesrdquo Journal of Virological Methods vol 203pp 23ndash32 2014

[28] G S Buzard D Baker M J Wolcott D A Norwood and L ADauphin ldquoMulti-platform comparison of ten commercial mas-ter mixes for probe-based real-time polymerase chain reactiondetection of bioterrorism threat agents for surge preparednessrdquoForensic Science International vol 223 no 1ndash3 pp 292ndash2972012

[29] A C Banyard J S Evans T R Luo and A R Fooks ldquoLyssavi-ruses and bats emergence and zoonotic threatrdquo Viruses vol 6no 8 pp 2974ndash2990 2014

[30] F Jacob R Guertler S Naim et al ldquoCareful selection ofreference genes is required for reliable performance of RT-qPCRin human normal and cancer cell linesrdquo PLoS ONE vol 8 no3 Article ID e59180 2013

[31] A Stahlberg M Kubista andM Pfaffl ldquoComparison of reversetranscriptases in gene expression analysisrdquo Clinical Chemistryvol 50 no 9 pp 1678ndash1680 2004

[32] M Fischer BHoffmannCM Freuling TMuller andM BeerldquoPerspectives on molecular detection methods of lyssavirusesrdquoBerliner und Munchener Tierarztliche Wochenschrift vol 125no 5-6 pp 264ndash271 2012

[33] M J Wacker and M P Godard ldquoAnalysis of one-step andtwo-step real-time RT-PCR using superscript IIIrdquo Journal ofBiomolecular Techniques vol 16 no 3 pp 266ndash271 2005

[34] D Schoder A Schmalwieser G Schauberger M Kuhn JHoorfar and M Wagner ldquoPhysical characteristics of six newthermocyclersrdquo Clinical Chemistry vol 49 no 6 pp 960ndash9632003

[35] D Schoder A Schmalwieser G Schauberger J Hoorfar MKuhn and M Wagner ldquoNovel approach for assessing perfor-mance of PCR cyclers used for diagnostic testingrdquo Journal ofClinical Microbiology vol 43 no 6 pp 2724ndash2728 2005

[36] H K Young I Yang Y-S Bae and S-R Park ldquoPerformanceevaluation of thermal cyclers for PCR in a rapid cyclingconditionrdquo BioTechniques vol 44 no 4 pp 495ndash505 2008

[37] World Organization for Animal Health ldquoPrinciples and meth-ods of validation of diagnostic assays for infectious diseasesrdquo inOIETerrestrialManualWorldOrganization for AnimalHealthParis France 2013

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


the 1980s and due to the immense versatility and sensitivityof PCR diagnostic methods based on PCR technology havebeen extensively applied to many infectious diseases [5]To date real-time PCR is the state-of-the-art technologyfor the quantification of nucleic acids Various nucleic aciddetection-based assays have thus been published for thedetection of Lyssavirus fragments using conventional gel-based PCR NASBA or real-time PCR [6] In Europe mostlaboratories working on rabies employ conventional andorreal-time PCR [7] due to their high specificity and sensitivityThe conventional methods are reported to be very sensitivewhen using a heminested RT-PCR [8] and compared to hem-inested RT-PCR real-time PCR is shown to be about 100ndash1000-fold more sensitive [9] However due to this high PCRsensitivity which engenders a risk of sample cross contamina-tion and therefore a risk of potential false positive results RT-PCR is not recommended by the World Health Organizationfor routine postmortem diagnosis of rabies in animals [4]

Real-time RT-PCR which provides rapid sensitive andspecific detection of rabies virus decreases the risk of samplecontamination Various real-time assays have thus beendescribed since 2004 [10] for the rapid detection of RABVor other Lyssavirus species [11 12] using the one-step method[13 14] or the two-step method [12 15 16] Two systems ofreal-time assays are widely used for the detection of RABVand lyssaviruses the SYBR Green real-time RT-PCR andthe TaqMan real-time RT-PCR which is to date the mostcommon technique implemented in the laboratories workingon rabies [17]

As real-time PCR assays have become routine in diagnos-tic laboratories and because of their prominence there hasbeen rapid growth of commercially available reagent systemsand detection platforms which greatly increase the optionsavailable to laboratories In this study we evaluated severalcommercial master mixes in order to identify those whichproduce the best results using one-step and two-step real-time RT-PCR for the determination of EBLV-1 Five differentsystems for the generation of cDNA five two-step PCR kitsand five one-step qRT-PCR kits were comparedThe analysedperformance evaluation criteria included the generation ofa standard curve reaction efficiency analytical sensitivityintra- and interassay repeatability as well as the costs and thepracticability of kits and thermocycling times The goal ofthis study was also to determine if the one-step and two-stepsystems had the same reaction efficiencies for the detectionof EBLV-1 and if one system was more sensitive than anotherusing two real-time thermocyclers

2 Materials and Methods

21 Rabies Virus Isolates and RNA Extraction The Europeanbat Lyssavirus type 1 virus (EBLV-1) was selected for this studyfor the generation of in vitroRNA transcriptThe virus EBLV-1 (ANSES strain numbered 05-09) was isolated in France in2000 from a rabid bat (Eptesicus serotinus) The isolate wasmouse passaged before RNA extraction

Viral RNAwas extracted from 200120583L of 10mouse braintissue suspension using the Iprep Pure Link Virus kit (Invit-rogen France) according to the manufacturerrsquos instructions

The RNA concentration was determined spectrophotometri-cally and the RNA was stored at minus70∘C until use

22 Preparation of In Vitro RNA Transcripts Briefly a one-step RT-PCR amplification was performed as previouslydescribed [8] on 5 120583L of extracted RNA (ie 250ndash500 ng)with specific rabies primers (JW12 (forward) 51015840-ATGTAA-CACCYCTACAATG and JW6 (reverse) 51015840-CARTTVGCR-CACATYTTRTG) The 606 bp PCR products were purifiedand inserted into a pGEM T easy vector (Promega France)according to the manufacturerrsquos instructions Recombinantplasmids were purified using the PerfectPrep EndoFreePlasmid Maxi Kit (5Prime France) and then concentratedusing a step of sodium acetateethanol precipitation and lin-earised with the PstI restriction enzyme (Promega France)The clones of PCR products were bidirectionally sequencedwith sense and antisense primers T7 and SP6 by BeckmanCoulter Genomics (Takeley United Kingdom) to confirm thepresence of the target insert

In vitro transcription was performed using T7 RNApolymerase according to the manufacturerrsquos instructions(MaxiScript SP6T7 kit Ambion France)The resulting RNAtranscripts were purified as recommended by the manufac-turer using 2120583L of DNAse I during 15min at 37∘C followedby a final purification step using the RNeasymini kit (QiagenFrance) The RNA concentration was determined by usingthe Qubit Fluorometer (Invitrogen France) RNA transcriptswere aliquoted to 107 copies120583L and then stored at minus70∘CTheRNAmolecule copy numberwas calculated following themanufacturerrsquos instructions (MaxiScript SP6T7 kit AmbionFrance) using the following formula 119884 molecules120583L =(119883 g120583L RNA[transcript length in nucleotides times 330] times6022sdot1023) The estimated number of copies was of 268 sdot 1011for EBLV-1 RNA transcript

The quality of RNA transcript was checked on serialtenfold dilutions (106 to 1 copie120583L) through measurementsof efficiency of amplification using a two-step SYBR GreenRT-PCR [12] PCR efficiency was of 99

23 Real-Time RT-PCR Assays

231 Primers Used Universal pan Lyssavirus primers wereselected for this study Two questionnaires respectivelyundertaken in 2011-2012 by the EURL laboratory havedemonstrated that 13 out of 21 National Reference Laborato-ries for Rabies (NRLs) used primers JW12 and N165-146 pre-viously described by Wakeley et al [18] These specific rabiesprimers (JW12 (forward) ATGTAACACCYCTACAATGand N165-146 (reverse) GCAGGGTAYTTRTACTCATA)were designed to detect the rabies virus N gene of all knownlyssaviruses [12]

232 Two-Step RT-qPCR Assays

(1) Commercial Master Mix Kits

(a) Reverse Transcription Kits (Table 1(a)) Five differentreverse transcription (RT) systems provided by five differentcompanies and five different qPCR kits provided by the same











Transcription Kit


+ random primer



water


Synthesis Kit











+ random primer


















Invitrogen


SupermixUniversal


















(c) Continued











Invitrogen
































3 Results








Table2Com

paris

onof

RT-qPC

RSybrGreen

kits(tw

o-ste

p)provided

bythes

amem

anufacturerRe

sults

offived

ifferentq

PCRkitson

arange

ofseventriplicated

ilutio

nsof

EBLV

-1RN

A

Testingof

different

RT-qPC

Rkits(tw

o-ste

p)provided

bythes

amem


Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

IScriptR

T+qP

CRSsoF

ast

QuantiTectR

T+qP

CRQuantitect

Maxim

aRT+qP

CRMaxim

aSS

ViloRT

+qP

CREx

press

qScriptR

T+qP

CRPerfe

cta

AverageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowast

106

5010

51658plusmn013

077

1947plusmn007

034

1650plusmn015

092

1501plusmn

005

035

1609plusmn003

019

1896plusmn004

021

2279plusmn026

115

1747plusmn009

052

1749plusmn010

054

1799plusmn029

159

105

5010

41993plusmn009

045

2289plusmn010

042

1950plusmn006

033

1821plusmn

005

026

1979plusmn003

013

2217plusmn006

025

2594plusmn009

035

2116plusmn029

138

2095plusmn003

015

2127plusmn015

072

104

5010

32328plusmn005

020

2589plusmn006

025

2295plusmn028

123

2168plusmn017

076

2318plusmn016

067

2575plusmn005

018

2960plusmn042

143

2429plusmn029

039

2440plusmn012

049

2455plusmn008

033

103

5010

22661plusmn

021

080

2964plusmn032

107

2643plusmn026

098

2481plusmn

005

019

2694plusmn008

030

2916plusmn023

080

3210plusmn019

060

2778plusmn030

109

2803plusmn074

263

2784plusmn012

042

102

5010

13045plusmn013

042

3220plusmn041

126

3059plusmn045

146

2833plusmn064

225

3140plusmn053

168

3238plusmn061

189

3512plusmn010

028

3046plusmn092

303

3105plusmn038

123

3135plusmn070

222

101

5010

023lowastlowast

13lowastlowast

23lowastlowast

3152plusmn13

3420

13lowastlowast

13lowastlowast

13lowastlowast

3276plusmn070

212

13lowastlowast

23lowastlowast

105

13lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

23lowastlowast

Efficiency

99

99

100

101

90

96

107

97

93

102

1198772

0999

0997

0997

0999

0999

099

9099

1099

7099

4099

8

119910-in

tercept

3665

3955

3631

3473

3767

3938

4183

3801

3850

3801

lowastCV

()=

standard


CVwas

notcalculated

whensomeo

fthe

parallelsfailedto

amplify


num

bero

fpositivesnu

mbero

fexp

erim

ents


erageC


deviation


Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)




















Table3Com

paris

onof

qRT-PC

RSY

BRGreen

(one-step)

kits

Results

offived

ifferentq

RT-PCR

kitson

arange

ofseventriplicated

ilutio

nsof

EBLV

-1in

vitro

RNAtranscrip

tsTestingof

different

qRT-PC

Rkits(one-step)

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Quantitect-1ste

pRo

torG

ene-1step

Verso-1step

SSIIIP

latinium-1ste

pqScript-1

step

AverageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowast

106

2010

61666plusmn008

045

1715plusmn005

029

1699plusmn006

038

1452plusmn019

032

1347plusmn003

019

1779plusmn024

132

1588plusmn022

136

1834plusmn032

172

1622plusmn004

022

1848plusmn004

020

105

2010

52006plusmn010

052

2062plusmn004

017

2040plusmn011

052

1790plusmn021

118

1655plusmn003

019

2113plusmn045

212

1942plusmn020

104

2160plusmn015

071

1964plusmn041

211

2238plusmn015

068

104

2010

42358plusmn014

058

2391plusmn

008

033

2382plusmn020

083

2143plusmn012

057

1986plusmn004

018

2460plusmn020

083

2287plusmn016

069

2517plusmn017

069

2308plusmn027

116

2611plusmn010

038

103

2010

32670plusmn024

091

2745plusmn028

101

2693plusmn003

011

2490plusmn023

092

2313plusmn016

067

2795plusmn026

093

2634plusmn033

124

2863plusmn030

106

2614plusmn010

038

2954plusmn025

086

102

2010

23042plusmn008

026

23lowastlowast

3019plusmn060

198

03lowastlowast

03lowastlowast

3198plusmn017

053

2977plusmn039

132

3174plusmn079

250

2893plusmn008

028

3107plusmn037

118

101

2010

13324plusmn029

087

13lowastlowast

3327plusmn15

3460

03lowastlowast

03lowastlowast

3434plusmn10

3299

3284plusmn044

133

23lowastlowast

2991plusmn

010

033

03lowastlowast

12

03lowastlowast

13lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

23lowastlowast

03lowastlowast

03lowastlowast

Efficiency

98

96

100

94

104

97

94

95

100

87

1198772

0998

0999

0999

0998

0999

099

6099

8099

7099

6099

8

119910-in

tercept

3689

3767

3699

3529

3278

3815

3680

3893

3893

4074

lowastCV

()=

standard


CVwas

notcalculated

whensomeo

fthe

parallelsfailedto

amplify


bero

fpositivesnu

mbero

fexp

erim

ents


erageC


deviation


Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)


Table4Detectio

nlim

itof

PCR(LOD95)for

two-ste

pandon

e-ste

pSY

BRGreen

RT-PCR

Two-ste

pRT

-qPC

RIScriptR

T+qP

CRSsoF

ast

BioR

adQuantiTectR

T+qP

CRQuantitect

QIAGEN

Maxim

aRT+qP

CRMaxim

aFE

RMEN

TAS

SSViloRT

+qP

CREx

press

INVITRO

GEN

qScriptR

T+qP

CRPerfe

cta

QUA

NTA

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

11055

171

8678

3941

2188

4488

44160

8076

3845

2267

34One-stepqR

T-PC

RQuantitect-1ste

pQIAGEN

RotorG

ene-1step

QIAGEN

Verso-1step

FERM

ENTA

SSSIIIP

latinium-1ste

pIN

VITRO

GEN

qScript-1

step

QUA

NTA

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

2040

497

994

127

254

321

641

406

812

2244

1530

101

202

50100

981

1962

lowastAllLO

D95results

werec

arrie

dou

tusin

gProb

itmod

ellowastAllPC

Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)


Table5Analysis

ofreprod

ucibilityof

one-ste

pandtwo-ste

pRT

-PCR

(a)

show

sfor

each

two-ste

pkittheintra-a

ndinterassay

reprod


nsof

EBLV

-1teste

deighttim

esin

each

ofthreeind

ependent

runs


nd(b)sho

wsthe


ucibilityfora

llteste

don

e-ste

pkits

(a)2-ste

pmetho

ds

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CV ()lowastlowast

AverageC

tplusmnSD

CtCV

()lowastlowast

IScriptR

T+qP

CRSsoF

ast

100

3046plusmn041

135

83008plusmn057

190

82982plusmn041

138

83012plusmn046

154

3012plusmn052

174

503111plusmn093

299

63184plusmn10

1319

63080plusmn046

151

83125plusmn080

256

3120plusmn088

283

253249plusmn059

183

63160plusmn067

212

83127plusmn068

219

63179plusmn065

204

3177plusmn080

251

103238plusmn091

281

63269plusmn119

363

63197plusmn076

239

53235plusmn095

294

3237plusmn097

299

53314

mdash1

mdashmdash

03198plusmn005

015

33256plusmn005

015

3227plusmn058

181

05

3217

mdash1

mdashmdash

03211plusmn076

236

33214plusmn076

236

3213plusmn062

193

250

3071plusmn

049

161

830

70plusmn039

128

830

61plusmn

033

108

830

67plusmn041

132

3067plusmn040

129

100

3230plusmn074

228

83203plusmn090

281

83219plusmn040

124

83217plusmn068

211

3217plusmn069

214

5034

02plusmn093

273

63324plusmn048

144

83316plusmn093

287

83347plusmn079

235

3342plusmn085

255

2534

63plusmn10

2295

83378plusmn075

223

634

24plusmn088

258

634

21plusmn

089

259

3425plusmn093

272

103595plusmn11

2312

53526plusmn047

134

534

68plusmn00

63

180

63530plusmn074

209

3526plusmn090

256

53384plusmn062

184

3mdash

mdash0

mdashmdash

034

83plusmn060

171

3384plusmn062

184

QuantiTectR

T+qP

CRQuantitect

250

3137plusmn069

220

83145plusmn052

165

83122plusmn046

147

83135plusmn056

177

3135plusmn055

175

100

3246plusmn080

246

73272plusmn092

281

73390plusmn090

265

73303plusmn087

264

3303plusmn10

5318

503359plusmn060

179

43434plusmn232

675

53366plusmn079

235

63386plusmn12

4363

3387plusmn14

0413

253298plusmn074

224

43358plusmn118

351

53412plusmn207

608

43356plusmn13

3394

3356plusmn13

8410

103354plusmn018

054

33386plusmn061

180

23473plusmn062

179

23404plusmn047

138

3397plusmn065

192

5mdash

mdash0

3552

mdash1

mdashmdash

03552

mdash3552

mdash50

034

09plusmn036

135

836

37plusmn118

326

63297plusmn038

116

834

30plusmn15

419

234

30plusmn15

4448

250

3642plusmn046

643

83366plusmn080

239

83361plusmn

083

246

834

56plusmn19

7376

3456plusmn19

7569

100

3439plusmn234

257

834

60plusmn098

284

834

50plusmn091

265

834

50plusmn089

269

3450plusmn089

258

5036

28plusmn088

475

63507plusmn091

259

734

92plusmn10

4297

73538plusmn13

2344

3538plusmn13

2374

253597plusmn17

2230

536

28plusmn088

242

436

44plusmn078

214

536

23plusmn079

229

3623plusmn079

217

10mdash

mdash0

mdashmdash

03765plusmn079

210

23765plusmn079

210

3765plusmn079

210


(a)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CV ()lowastlowast

AverageC

tplusmnSD

CtCV

()lowastlowast

Maxim

aRT

+qP

CRMaxim

a

100

3183plusmn084

263

3138plusmn022

070

3009plusmn041

137

311plusmn049

157

311plusmn092

295

503335plusmn10

9326

83342plusmn088

264

53112plusmn064

205

83263plusmn087

265

3251plusmn

140

432

253367plusmn088

261

53277plusmn112

340

63247plusmn072

221

73297plusmn090

274

3290plusmn099

302

103716plusmn464

1249

33400plusmn14

1416

43435plusmn13

3388

53517plusmn246

684

3494plusmn263

754

53544plusmn024

068

23865

mdash1

3458plusmn12

4357

33622plusmn074

212

3555plusmn17

6496

25

mdashmdash

03314

mdash1

3347plusmn17

6526

23330plusmn17

6526

3336plusmn12

6377

250

2928plusmn058

199

829

05plusmn038

130

829

04plusmn039

135

829

12plusmn045

155

2912plusmn045

156

100

3053plusmn056

185

830

17plusmn048

159

830

31plusmn

043

143

830

34plusmn050

162

3034plusmn050

164

503154plusmn12

5397

83188plusmn13

7429

83172plusmn056

177

83171plusmn

108

334

3171plusmn

108

340

253300plusmn115

348

63238plusmn079

243

73238plusmn082

254

73256plusmn092

282

3256plusmn092

281

103308plusmn064

193

33302plusmn13

8417

43340plusmn14

6438

43317plusmn115

349

3317plusmn11

5347

503303plusmn015

045

23355

mdash1

3299

mdash1

3315plusmn028

045

3315plusmn028

085

qScriptR

T+qP

CRPerfe

cta

250

2704plusmn024

088

82675plusmn026

098

82677plusmn024

090

82685plusmn025

092

2685plusmn027

101

100

3054plusmn054

176

82984plusmn054

182

83046plusmn10

7350

83028plusmn072

236

3028plusmn079

261

503203plusmn290

905

73072plusmn089

292

83078plusmn082

267

83118plusmn15

4488

3114plusmn17

7568

253250plusmn098

302

73241plusmn

133

410

73363plusmn216

642

53285plusmn14

9452

3276plusmn14

9456

103222plusmn081

252

53287plusmn046

139

33270plusmn056

171

23260plusmn061

187

3251plusmn

069

211

53278plusmn015

045

2mdash

mdash0

3356plusmn15

5461

23318plusmn085

253

3318plusmn10

0303

250

2947plusmn022

074

829

49plusmn054

183

729

82plusmn038

129

829

60plusmn041

129

2960plusmn041

139

100

3099plusmn10

9352

83114plusmn12

6404

83102plusmn052

169

83105plusmn096

308

3105plusmn096

311

503187plusmn060

188

83137plusmn057

180

83313plusmn11

2337

83212plusmn10

8235

3212plusmn10

8335

253335plusmn299

896

73249plusmn068

211

83271plusmn

087

265

83283plusmn17

2457

3283plusmn17

2524

103300plusmn049

147

53345plusmn018

053

23298plusmn074

225

43307plusmn054

142

3307plusmn054

165

53304plusmn10

1306

23303plusmn074

223

23359

mdash1

3314plusmn067

265

3314plusmn067

203


(a)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CV ()lowastlowast

AverageC

tplusmnSD

CtCV

()lowastlowast

SSViloRT

+qP

CREx

press

100

2768plusmn059

213

82782plusmn055

197

82759plusmn029

106

82770plusmn048

172

2770plusmn048

174

502942plusmn062

211

82927plusmn054

185

82921plusmn

060

206

82930plusmn059

201

2930plusmn057

195

253038plusmn067

221

63022plusmn12

4411

82960plusmn048

162

83007plusmn080

264

3004plusmn090

300

103136plusmn049

158

73130plusmn071

227

73192plusmn078

245

63153plusmn066

210

3151plusmn

069

219

53149plusmn053

168

43163plusmn098

308

43164

mdash1

3159plusmn075

238

3157plusmn068

216

05

mdashmdash

0mdash

mdash0

3159

mdash1

3159

mdash3159

mdash100

3178plusmn053

167

830

13plusmn032

107

830

75plusmn065

211

830

89plusmn085

162

3089plusmn085

276

503296plusmn042

129

83134plusmn050

161

83149plusmn097

307

83193plusmn099

199

3193plusmn099

309

253372plusmn10

5310

73215plusmn098

306

83209plusmn022

069

63266plusmn113

228

3266plusmn11

3346

1034

44plusmn060

174

73361plusmn

046

135

53330plusmn043

129

63383plusmn071

146

3383plusmn071

209

53521plusmn

053

151

73264plusmn036

111

53299plusmn006

017

23397plusmn13

5093

3397plusmn13

5398

25

3956plusmn18

6471

334

35

13355plusmn057

169

336

24plusmn332

320

3624plusmn332

916


varia

bilitya

ndcoeffi

ciento

fvariatio

nswerec

alculated


CV=sta



100intra-assayC

V=mean

oftheC

Vso

fallruns

lowastAv

erageC


deviation

lowastlowastCV

()=

standard



Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)

(b)1stepmetho

ds

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CVlowastlowast

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Quantitect1step

502976plusmn064

216

82894plusmn055

189

82927plusmn023

079

82932plusmn047

161

2932plusmn059

106

253090plusmn088

286

82984plusmn10

9365

83098plusmn076

245

83057plusmn091

299

3057plusmn10

3202

103172plusmn10

4326

73205plusmn10

6332

73230plusmn061

188

83203plusmn090

282

3204plusmn090

336

53334plusmn12

0360

63238plusmn074

228

73285plusmn084

255

73286plusmn092

281

3283plusmn096

281

25

3251plusmn

060

185

43390plusmn033

096

43445plusmn15

2442

53362plusmn082

241

3368plusmn12

7377

13338plusmn096

288

43203plusmn026

080

33292plusmn047

143

53277plusmn056

170

3285plusmn079

242

5030

92plusmn047

152

83123plusmn12

9040

83111plusmn13

2041

83108plusmn13

8043

3108plusmn043

138

253177plusmn073

229

83192plusmn14

9048

83191plusmn

093

030

83187plusmn15

7050

3187plusmn051

161

103260plusmn047

144

83281plusmn

061

187

53249plusmn065

199

83263plusmn058

177

3263plusmn057

175

53369plusmn085

253

73382plusmn291

098

53328plusmn250

083

63360plusmn265

089

3357plusmn086

257

25

3436plusmn073

214

53365plusmn18

8063

23327plusmn18

8062

33376plusmn19

6066

3380plusmn080

236

134

55plusmn17

7513

334

97plusmn117

041

53392plusmn341

116

134

48plusmn324

111

3445plusmn13

7397


(b)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CVlowastlowast

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

RotorG

ene1

step

1000

2640plusmn045

169

82374plusmn040

167

82701plusmn

118

438

82572plusmn16

2258

2572plusmn16

2632

500

2749plusmn046

166

82429plusmn034

139

82742plusmn10

4378

82640plusmn16

6228

2640plusmn16

6628

250

2873plusmn076

265

82490plusmn049

196

82927plusmn060

204

72756plusmn209

222

2756plusmn209

758

100

3070plusmn021

067

32550

mdash1

3119plusmn066

211

23000plusmn224

139

3000plusmn224

746

503031

mdash1

mdashmdash

0mdash

mdash0

3031

mdash3031

mdash25

3112plusmn050

161

2mdash

mdash0

mdashmdash

03212plusmn050

161

3212plusmn050

161

5028

87plusmn099

341

829

25plusmn074

252

82797plusmn065

233

828

70plusmn079

276

2870plusmn094

329

2529

49plusmn10

1344

830

36plusmn070

229

828

57plusmn039

136

829

47plusmn070

236

2947plusmn10

3350

1030

29plusmn12

0398

830

61plusmn

144

470

828

76plusmn040

139

829

89plusmn10

1336

2989plusmn13

4450

530

82plusmn10

6343

63147plusmn13

0412

629

90plusmn040

135

830

73plusmn092

297

3065plusmn11

2366

25

3087plusmn114

368

63169plusmn057

181

529

10plusmn037

126

530

55plusmn069

225

3057plusmn13

1429

13122

mdash1

3153

mdash1

3201plusmn

044

137

53159plusmn044

137

3183plusmn048

151

Verso1step

500

2650plusmn018

068

82656plusmn014

053

82679plusmn023

085

82661plusmn

018

069

2661plusmn

022

082

250

2743plusmn018

065

82796plusmn027

098

82789plusmn036

127

82776plusmn027

097

2776plusmn036

130

100

2894plusmn050

172

82942plusmn053

180

82991plusmn

118

396

82943plusmn074

249

2943plusmn087

295

503049plusmn044

143

73138plusmn086

274

83129plusmn15

2487

83105plusmn094

301

3108plusmn10

9350

253282plusmn114

346

73290plusmn052

157

53250plusmn092

282

73274plusmn086

262

3272plusmn090

274

103342plusmn097

291

33522plusmn10

5299

23442

mdash1

3435plusmn10

1295

3419plusmn118

344

250

2939plusmn048

164

829

40plusmn023

079

829

10plusmn053

183

829

20plusmn042

142

2930plusmn044

150

100

3055plusmn025

080

830

32plusmn038

125

830

02plusmn024

082

630

29plusmn029

096

3032plusmn036

119

503116plusmn050

159

83110plusmn052

168

830

87plusmn042

135

73104plusmn048

154

3105plusmn048

153

253171plusmn

032

102

73161plusmn

042

134

83135plusmn058

184

83156plusmn044

140

3155plusmn046

147

103186plusmn054

169

73176plusmn029

091

73165plusmn064

201

43176plusmn049

154

3177plusmn046

145

53309plusmn098

297

53313

13343plusmn074

223

43322plusmn086

260

3326plusmn080

239


(b)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CVlowastlowast

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

qScript1

step

1000

2324plusmn012

053

82297plusmn024

103

82321plusmn

009

037

82314plusmn015

064

2314plusmn020

086

750

2364plusmn021

088

82329plusmn008

035

82352plusmn028

121

82348plusmn019

081

2348plusmn025

106

500

2446plusmn032

132

82406plusmn024

098

82407plusmn029

122

82419plusmn028

117

2419plusmn033

137

250

2538plusmn027

107

82493plusmn026

105

82514plusmn034

137

82515plusmn029

116

2515plusmn034

135

100

2626plusmn031

116

82579plusmn024

093

72595plusmn047

180

52600plusmn034

130

2602plusmn038

146

50mdash

mdash0

mdashmdash

02645plusmn018

070

22645plusmn018

070

2645plusmn018

070

2500

2813plusmn083

294

82778plusmn045

160

826

32plusmn030

113

82741plusmn

052

189

2741plusmn

097

353

1000

2878plusmn009

033

829

40plusmn15

8539

824

89plusmn030

122

82769plusmn066

231

2769plusmn223

805

750

2908plusmn024

082

828

92plusmn025

085

828

75plusmn011

038

828

92plusmn020

068

2892plusmn024

084

500

3069plusmn048

157

729

76plusmn055

186

82763plusmn056

201

529

36plusmn053

181

2966plusmn12

6424

250

3122plusmn023

073

730

05plusmn037

124

8mdash

mdash0

3064plusmn030

099

3060plusmn067

220

100

mdashmdash

030

00

mdash1

mdashmdash

030

00

mdash30

00

mdash

SSIIIP

latinium

1step

1000

2479plusmn024

097

82381plusmn

021

089

82446plusmn015

060

82435plusmn020

082

2435plusmn046

188

750

2518plusmn024

095

82430plusmn012

051

82497plusmn010

042

82482plusmn016

063

2482plusmn041

167

500

2666plusmn082

307

82536plusmn023

092

82619plusmn024

092

82607plusmn043

164

2607plusmn073

282

250

2866plusmn086

301

82838plusmn19

1672

82897plusmn059

205

82867plusmn112

393

2867plusmn12

2427

100

2917plusmn053

180

82778plusmn049

175

82816plusmn053

189

72837plusmn052

182

2838plusmn078

276

503095plusmn043

139

22911plusmn067

231

42993plusmn111

370

33000plusmn074

247

2979plusmn10

3347

250

2627plusmn026

099

826

70plusmn027

102

826

67plusmn042

156

826

55plusmn037

119

2655plusmn037

139

100

2722plusmn068

248

828

02plusmn039

138

82774plusmn034

121

82766plusmn058

169

2766plusmn058

209

502756plusmn053

194

828

26plusmn041

147

828

33plusmn040

141

828

05plusmn056

161

2805plusmn056

200

252787plusmn069

247

829

34plusmn055

189

828

58plusmn041

145

828

59plusmn082

194

2859plusmn082

285

1028

75plusmn078

270

629

44plusmn055

186

529

08plusmn033

114

729

07plusmn060

190

2907plusmn060

207

528

82plusmn015

053

428

71plusmn

021

072

328

88plusmn032

112

428

81plusmn

023

079

2881plusmn

023

079

Theintra-a

ndinterassay

varia

bilityandcoeffi

ciento

fvariatio

nswerec

alculated


=standard

deviationof



100intra-assayCV

=mean

oftheC

Vso

fallruns

lowastAv

erageC


deviation

lowastlowastCV

()=

standard



Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)






RotorGene QMDx

Mx3005P






4 Discussion












5 Conclusion



Abbreviations




Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology











Transcription Kit


+ random primer



water


Synthesis Kit











+ random primer


















Invitrogen


SupermixUniversal


















(c) Continued











Invitrogen
































3 Results








Table2Com

paris

onof

RT-qPC

RSybrGreen

kits(tw

o-ste

p)provided

bythes

amem

anufacturerRe

sults

offived

ifferentq

PCRkitson

arange

ofseventriplicated

ilutio

nsof

EBLV

-1RN

A

Testingof

different

RT-qPC

Rkits(tw

o-ste

p)provided

bythes

amem


Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

IScriptR

T+qP

CRSsoF

ast

QuantiTectR

T+qP

CRQuantitect

Maxim

aRT+qP

CRMaxim

aSS

ViloRT

+qP

CREx

press

qScriptR

T+qP

CRPerfe

cta

AverageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowast

106

5010

51658plusmn013

077

1947plusmn007

034

1650plusmn015

092

1501plusmn

005

035

1609plusmn003

019

1896plusmn004

021

2279plusmn026

115

1747plusmn009

052

1749plusmn010

054

1799plusmn029

159

105

5010

41993plusmn009

045

2289plusmn010

042

1950plusmn006

033

1821plusmn

005

026

1979plusmn003

013

2217plusmn006

025

2594plusmn009

035

2116plusmn029

138

2095plusmn003

015

2127plusmn015

072

104

5010

32328plusmn005

020

2589plusmn006

025

2295plusmn028

123

2168plusmn017

076

2318plusmn016

067

2575plusmn005

018

2960plusmn042

143

2429plusmn029

039

2440plusmn012

049

2455plusmn008

033

103

5010

22661plusmn

021

080

2964plusmn032

107

2643plusmn026

098

2481plusmn

005

019

2694plusmn008

030

2916plusmn023

080

3210plusmn019

060

2778plusmn030

109

2803plusmn074

263

2784plusmn012

042

102

5010

13045plusmn013

042

3220plusmn041

126

3059plusmn045

146

2833plusmn064

225

3140plusmn053

168

3238plusmn061

189

3512plusmn010

028

3046plusmn092

303

3105plusmn038

123

3135plusmn070

222

101

5010

023lowastlowast

13lowastlowast

23lowastlowast

3152plusmn13

3420

13lowastlowast

13lowastlowast

13lowastlowast

3276plusmn070

212

13lowastlowast

23lowastlowast

105

13lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

23lowastlowast

Efficiency

99

99

100

101

90

96

107

97

93

102

1198772

0999

0997

0997

0999

0999

099

9099

1099

7099

4099

8

119910-in

tercept

3665

3955

3631

3473

3767

3938

4183

3801

3850

3801

lowastCV

()=

standard


CVwas

notcalculated

whensomeo

fthe

parallelsfailedto

amplify


num

bero

fpositivesnu

mbero

fexp

erim

ents


erageC


deviation


Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)




















Table3Com

paris

onof

qRT-PC

RSY

BRGreen

(one-step)

kits

Results

offived

ifferentq

RT-PCR

kitson

arange

ofseventriplicated

ilutio

nsof

EBLV

-1in

vitro

RNAtranscrip

tsTestingof

different

qRT-PC

Rkits(one-step)

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Quantitect-1ste

pRo

torG

ene-1step

Verso-1step

SSIIIP

latinium-1ste

pqScript-1

step

AverageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowast

106

2010

61666plusmn008

045

1715plusmn005

029

1699plusmn006

038

1452plusmn019

032

1347plusmn003

019

1779plusmn024

132

1588plusmn022

136

1834plusmn032

172

1622plusmn004

022

1848plusmn004

020

105

2010

52006plusmn010

052

2062plusmn004

017

2040plusmn011

052

1790plusmn021

118

1655plusmn003

019

2113plusmn045

212

1942plusmn020

104

2160plusmn015

071

1964plusmn041

211

2238plusmn015

068

104

2010

42358plusmn014

058

2391plusmn

008

033

2382plusmn020

083

2143plusmn012

057

1986plusmn004

018

2460plusmn020

083

2287plusmn016

069

2517plusmn017

069

2308plusmn027

116

2611plusmn010

038

103

2010

32670plusmn024

091

2745plusmn028

101

2693plusmn003

011

2490plusmn023

092

2313plusmn016

067

2795plusmn026

093

2634plusmn033

124

2863plusmn030

106

2614plusmn010

038

2954plusmn025

086

102

2010

23042plusmn008

026

23lowastlowast

3019plusmn060

198

03lowastlowast

03lowastlowast

3198plusmn017

053

2977plusmn039

132

3174plusmn079

250

2893plusmn008

028

3107plusmn037

118

101

2010

13324plusmn029

087

13lowastlowast

3327plusmn15

3460

03lowastlowast

03lowastlowast

3434plusmn10

3299

3284plusmn044

133

23lowastlowast

2991plusmn

010

033

03lowastlowast

12

03lowastlowast

13lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

23lowastlowast

03lowastlowast

03lowastlowast

Efficiency

98

96

100

94

104

97

94

95

100

87

1198772

0998

0999

0999

0998

0999

099

6099

8099

7099

6099

8

119910-in

tercept

3689

3767

3699

3529

3278

3815

3680

3893

3893

4074

lowastCV

()=

standard


CVwas

notcalculated

whensomeo

fthe

parallelsfailedto

amplify


bero

fpositivesnu

mbero

fexp

erim

ents


erageC


deviation


Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)


Table4Detectio

nlim

itof

PCR(LOD95)for

two-ste

pandon

e-ste

pSY

BRGreen

RT-PCR

Two-ste

pRT

-qPC

RIScriptR

T+qP

CRSsoF

ast

BioR

adQuantiTectR

T+qP

CRQuantitect

QIAGEN

Maxim

aRT+qP

CRMaxim

aFE

RMEN

TAS

SSViloRT

+qP

CREx

press

INVITRO

GEN

qScriptR

T+qP

CRPerfe

cta

QUA

NTA

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

11055

171

8678

3941

2188

4488

44160

8076

3845

2267

34One-stepqR

T-PC

RQuantitect-1ste

pQIAGEN

RotorG

ene-1step

QIAGEN

Verso-1step

FERM

ENTA

SSSIIIP

latinium-1ste

pIN

VITRO

GEN

qScript-1

step

QUA

NTA

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

2040

497

994

127

254

321

641

406

812

2244

1530

101

202

50100

981

1962

lowastAllLO

D95results

werec

arrie

dou

tusin

gProb

itmod

ellowastAllPC

Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)


Table5Analysis

ofreprod

ucibilityof

one-ste

pandtwo-ste

pRT

-PCR

(a)

show

sfor

each

two-ste

pkittheintra-a

ndinterassay

reprod


nsof

EBLV

-1teste

deighttim

esin

each

ofthreeind

ependent

runs


nd(b)sho

wsthe


ucibilityfora

llteste

don

e-ste

pkits

(a)2-ste

pmetho

ds

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CV ()lowastlowast

AverageC

tplusmnSD

CtCV

()lowastlowast

IScriptR

T+qP

CRSsoF

ast

100

3046plusmn041

135

83008plusmn057

190

82982plusmn041

138

83012plusmn046

154

3012plusmn052

174

503111plusmn093

299

63184plusmn10

1319

63080plusmn046

151

83125plusmn080

256

3120plusmn088

283

253249plusmn059

183

63160plusmn067

212

83127plusmn068

219

63179plusmn065

204

3177plusmn080

251

103238plusmn091

281

63269plusmn119

363

63197plusmn076

239

53235plusmn095

294

3237plusmn097

299

53314

mdash1

mdashmdash

03198plusmn005

015

33256plusmn005

015

3227plusmn058

181

05

3217

mdash1

mdashmdash

03211plusmn076

236

33214plusmn076

236

3213plusmn062

193

250

3071plusmn

049

161

830

70plusmn039

128

830

61plusmn

033

108

830

67plusmn041

132

3067plusmn040

129

100

3230plusmn074

228

83203plusmn090

281

83219plusmn040

124

83217plusmn068

211

3217plusmn069

214

5034

02plusmn093

273

63324plusmn048

144

83316plusmn093

287

83347plusmn079

235

3342plusmn085

255

2534

63plusmn10

2295

83378plusmn075

223

634

24plusmn088

258

634

21plusmn

089

259

3425plusmn093

272

103595plusmn11

2312

53526plusmn047

134

534

68plusmn00

63

180

63530plusmn074

209

3526plusmn090

256

53384plusmn062

184

3mdash

mdash0

mdashmdash

034

83plusmn060

171

3384plusmn062

184

QuantiTectR

T+qP

CRQuantitect

250

3137plusmn069

220

83145plusmn052

165

83122plusmn046

147

83135plusmn056

177

3135plusmn055

175

100

3246plusmn080

246

73272plusmn092

281

73390plusmn090

265

73303plusmn087

264

3303plusmn10

5318

503359plusmn060

179

43434plusmn232

675

53366plusmn079

235

63386plusmn12

4363

3387plusmn14

0413

253298plusmn074

224

43358plusmn118

351

53412plusmn207

608

43356plusmn13

3394

3356plusmn13

8410

103354plusmn018

054

33386plusmn061

180

23473plusmn062

179

23404plusmn047

138

3397plusmn065

192

5mdash

mdash0

3552

mdash1

mdashmdash

03552

mdash3552

mdash50

034

09plusmn036

135

836

37plusmn118

326

63297plusmn038

116

834

30plusmn15

419

234

30plusmn15

4448

250

3642plusmn046

643

83366plusmn080

239

83361plusmn

083

246

834

56plusmn19

7376

3456plusmn19

7569

100

3439plusmn234

257

834

60plusmn098

284

834

50plusmn091

265

834

50plusmn089

269

3450plusmn089

258

5036

28plusmn088

475

63507plusmn091

259

734

92plusmn10

4297

73538plusmn13

2344

3538plusmn13

2374

253597plusmn17

2230

536

28plusmn088

242

436

44plusmn078

214

536

23plusmn079

229

3623plusmn079

217

10mdash

mdash0

mdashmdash

03765plusmn079

210

23765plusmn079

210

3765plusmn079

210


(a)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CV ()lowastlowast

AverageC

tplusmnSD

CtCV

()lowastlowast

Maxim

aRT

+qP

CRMaxim

a

100

3183plusmn084

263

3138plusmn022

070

3009plusmn041

137

311plusmn049

157

311plusmn092

295

503335plusmn10

9326

83342plusmn088

264

53112plusmn064

205

83263plusmn087

265

3251plusmn

140

432

253367plusmn088

261

53277plusmn112

340

63247plusmn072

221

73297plusmn090

274

3290plusmn099

302

103716plusmn464

1249

33400plusmn14

1416

43435plusmn13

3388

53517plusmn246

684

3494plusmn263

754

53544plusmn024

068

23865

mdash1

3458plusmn12

4357

33622plusmn074

212

3555plusmn17

6496

25

mdashmdash

03314

mdash1

3347plusmn17

6526

23330plusmn17

6526

3336plusmn12

6377

250

2928plusmn058

199

829

05plusmn038

130

829

04plusmn039

135

829

12plusmn045

155

2912plusmn045

156

100

3053plusmn056

185

830

17plusmn048

159

830

31plusmn

043

143

830

34plusmn050

162

3034plusmn050

164

503154plusmn12

5397

83188plusmn13

7429

83172plusmn056

177

83171plusmn

108

334

3171plusmn

108

340

253300plusmn115

348

63238plusmn079

243

73238plusmn082

254

73256plusmn092

282

3256plusmn092

281

103308plusmn064

193

33302plusmn13

8417

43340plusmn14

6438

43317plusmn115

349

3317plusmn11

5347

503303plusmn015

045

23355

mdash1

3299

mdash1

3315plusmn028

045

3315plusmn028

085

qScriptR

T+qP

CRPerfe

cta

250

2704plusmn024

088

82675plusmn026

098

82677plusmn024

090

82685plusmn025

092

2685plusmn027

101

100

3054plusmn054

176

82984plusmn054

182

83046plusmn10

7350

83028plusmn072

236

3028plusmn079

261

503203plusmn290

905

73072plusmn089

292

83078plusmn082

267

83118plusmn15

4488

3114plusmn17

7568

253250plusmn098

302

73241plusmn

133

410

73363plusmn216

642

53285plusmn14

9452

3276plusmn14

9456

103222plusmn081

252

53287plusmn046

139

33270plusmn056

171

23260plusmn061

187

3251plusmn

069

211

53278plusmn015

045

2mdash

mdash0

3356plusmn15

5461

23318plusmn085

253

3318plusmn10

0303

250

2947plusmn022

074

829

49plusmn054

183

729

82plusmn038

129

829

60plusmn041

129

2960plusmn041

139

100

3099plusmn10

9352

83114plusmn12

6404

83102plusmn052

169

83105plusmn096

308

3105plusmn096

311

503187plusmn060

188

83137plusmn057

180

83313plusmn11

2337

83212plusmn10

8235

3212plusmn10

8335

253335plusmn299

896

73249plusmn068

211

83271plusmn

087

265

83283plusmn17

2457

3283plusmn17

2524

103300plusmn049

147

53345plusmn018

053

23298plusmn074

225

43307plusmn054

142

3307plusmn054

165

53304plusmn10

1306

23303plusmn074

223

23359

mdash1

3314plusmn067

265

3314plusmn067

203


(a)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CV ()lowastlowast

AverageC

tplusmnSD

CtCV

()lowastlowast

SSViloRT

+qP

CREx

press

100

2768plusmn059

213

82782plusmn055

197

82759plusmn029

106

82770plusmn048

172

2770plusmn048

174

502942plusmn062

211

82927plusmn054

185

82921plusmn

060

206

82930plusmn059

201

2930plusmn057

195

253038plusmn067

221

63022plusmn12

4411

82960plusmn048

162

83007plusmn080

264

3004plusmn090

300

103136plusmn049

158

73130plusmn071

227

73192plusmn078

245

63153plusmn066

210

3151plusmn

069

219

53149plusmn053

168

43163plusmn098

308

43164

mdash1

3159plusmn075

238

3157plusmn068

216

05

mdashmdash

0mdash

mdash0

3159

mdash1

3159

mdash3159

mdash100

3178plusmn053

167

830

13plusmn032

107

830

75plusmn065

211

830

89plusmn085

162

3089plusmn085

276

503296plusmn042

129

83134plusmn050

161

83149plusmn097

307

83193plusmn099

199

3193plusmn099

309

253372plusmn10

5310

73215plusmn098

306

83209plusmn022

069

63266plusmn113

228

3266plusmn11

3346

1034

44plusmn060

174

73361plusmn

046

135

53330plusmn043

129

63383plusmn071

146

3383plusmn071

209

53521plusmn

053

151

73264plusmn036

111

53299plusmn006

017

23397plusmn13

5093

3397plusmn13

5398

25

3956plusmn18

6471

334

35

13355plusmn057

169

336

24plusmn332

320

3624plusmn332

916


varia

bilitya

ndcoeffi

ciento

fvariatio

nswerec

alculated


CV=sta



100intra-assayC

V=mean

oftheC

Vso

fallruns

lowastAv

erageC


deviation

lowastlowastCV

()=

standard



Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)

(b)1stepmetho

ds

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CVlowastlowast

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Quantitect1step

502976plusmn064

216

82894plusmn055

189

82927plusmn023

079

82932plusmn047

161

2932plusmn059

106

253090plusmn088

286

82984plusmn10

9365

83098plusmn076

245

83057plusmn091

299

3057plusmn10

3202

103172plusmn10

4326

73205plusmn10

6332

73230plusmn061

188

83203plusmn090

282

3204plusmn090

336

53334plusmn12

0360

63238plusmn074

228

73285plusmn084

255

73286plusmn092

281

3283plusmn096

281

25

3251plusmn

060

185

43390plusmn033

096

43445plusmn15

2442

53362plusmn082

241

3368plusmn12

7377

13338plusmn096

288

43203plusmn026

080

33292plusmn047

143

53277plusmn056

170

3285plusmn079

242

5030

92plusmn047

152

83123plusmn12

9040

83111plusmn13

2041

83108plusmn13

8043

3108plusmn043

138

253177plusmn073

229

83192plusmn14

9048

83191plusmn

093

030

83187plusmn15

7050

3187plusmn051

161

103260plusmn047

144

83281plusmn

061

187

53249plusmn065

199

83263plusmn058

177

3263plusmn057

175

53369plusmn085

253

73382plusmn291

098

53328plusmn250

083

63360plusmn265

089

3357plusmn086

257

25

3436plusmn073

214

53365plusmn18

8063

23327plusmn18

8062

33376plusmn19

6066

3380plusmn080

236

134

55plusmn17

7513

334

97plusmn117

041

53392plusmn341

116

134

48plusmn324

111

3445plusmn13

7397


(b)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CVlowastlowast

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

RotorG

ene1

step

1000

2640plusmn045

169

82374plusmn040

167

82701plusmn

118

438

82572plusmn16

2258

2572plusmn16

2632

500

2749plusmn046

166

82429plusmn034

139

82742plusmn10

4378

82640plusmn16

6228

2640plusmn16

6628

250

2873plusmn076

265

82490plusmn049

196

82927plusmn060

204

72756plusmn209

222

2756plusmn209

758

100

3070plusmn021

067

32550

mdash1

3119plusmn066

211

23000plusmn224

139

3000plusmn224

746

503031

mdash1

mdashmdash

0mdash

mdash0

3031

mdash3031

mdash25

3112plusmn050

161

2mdash

mdash0

mdashmdash

03212plusmn050

161

3212plusmn050

161

5028

87plusmn099

341

829

25plusmn074

252

82797plusmn065

233

828

70plusmn079

276

2870plusmn094

329

2529

49plusmn10

1344

830

36plusmn070

229

828

57plusmn039

136

829

47plusmn070

236

2947plusmn10

3350

1030

29plusmn12

0398

830

61plusmn

144

470

828

76plusmn040

139

829

89plusmn10

1336

2989plusmn13

4450

530

82plusmn10

6343

63147plusmn13

0412

629

90plusmn040

135

830

73plusmn092

297

3065plusmn11

2366

25

3087plusmn114

368

63169plusmn057

181

529

10plusmn037

126

530

55plusmn069

225

3057plusmn13

1429

13122

mdash1

3153

mdash1

3201plusmn

044

137

53159plusmn044

137

3183plusmn048

151

Verso1step

500

2650plusmn018

068

82656plusmn014

053

82679plusmn023

085

82661plusmn

018

069

2661plusmn

022

082

250

2743plusmn018

065

82796plusmn027

098

82789plusmn036

127

82776plusmn027

097

2776plusmn036

130

100

2894plusmn050

172

82942plusmn053

180

82991plusmn

118

396

82943plusmn074

249

2943plusmn087

295

503049plusmn044

143

73138plusmn086

274

83129plusmn15

2487

83105plusmn094

301

3108plusmn10

9350

253282plusmn114

346

73290plusmn052

157

53250plusmn092

282

73274plusmn086

262

3272plusmn090

274

103342plusmn097

291

33522plusmn10

5299

23442

mdash1

3435plusmn10

1295

3419plusmn118

344

250

2939plusmn048

164

829

40plusmn023

079

829

10plusmn053

183

829

20plusmn042

142

2930plusmn044

150

100

3055plusmn025

080

830

32plusmn038

125

830

02plusmn024

082

630

29plusmn029

096

3032plusmn036

119

503116plusmn050

159

83110plusmn052

168

830

87plusmn042

135

73104plusmn048

154

3105plusmn048

153

253171plusmn

032

102

73161plusmn

042

134

83135plusmn058

184

83156plusmn044

140

3155plusmn046

147

103186plusmn054

169

73176plusmn029

091

73165plusmn064

201

43176plusmn049

154

3177plusmn046

145

53309plusmn098

297

53313

13343plusmn074

223

43322plusmn086

260

3326plusmn080

239


(b)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CVlowastlowast

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

qScript1

step

1000

2324plusmn012

053

82297plusmn024

103

82321plusmn

009

037

82314plusmn015

064

2314plusmn020

086

750

2364plusmn021

088

82329plusmn008

035

82352plusmn028

121

82348plusmn019

081

2348plusmn025

106

500

2446plusmn032

132

82406plusmn024

098

82407plusmn029

122

82419plusmn028

117

2419plusmn033

137

250

2538plusmn027

107

82493plusmn026

105

82514plusmn034

137

82515plusmn029

116

2515plusmn034

135

100

2626plusmn031

116

82579plusmn024

093

72595plusmn047

180

52600plusmn034

130

2602plusmn038

146

50mdash

mdash0

mdashmdash

02645plusmn018

070

22645plusmn018

070

2645plusmn018

070

2500

2813plusmn083

294

82778plusmn045

160

826

32plusmn030

113

82741plusmn

052

189

2741plusmn

097

353

1000

2878plusmn009

033

829

40plusmn15

8539

824

89plusmn030

122

82769plusmn066

231

2769plusmn223

805

750

2908plusmn024

082

828

92plusmn025

085

828

75plusmn011

038

828

92plusmn020

068

2892plusmn024

084

500

3069plusmn048

157

729

76plusmn055

186

82763plusmn056

201

529

36plusmn053

181

2966plusmn12

6424

250

3122plusmn023

073

730

05plusmn037

124

8mdash

mdash0

3064plusmn030

099

3060plusmn067

220

100

mdashmdash

030

00

mdash1

mdashmdash

030

00

mdash30

00

mdash

SSIIIP

latinium

1step

1000

2479plusmn024

097

82381plusmn

021

089

82446plusmn015

060

82435plusmn020

082

2435plusmn046

188

750

2518plusmn024

095

82430plusmn012

051

82497plusmn010

042

82482plusmn016

063

2482plusmn041

167

500

2666plusmn082

307

82536plusmn023

092

82619plusmn024

092

82607plusmn043

164

2607plusmn073

282

250

2866plusmn086

301

82838plusmn19

1672

82897plusmn059

205

82867plusmn112

393

2867plusmn12

2427

100

2917plusmn053

180

82778plusmn049

175

82816plusmn053

189

72837plusmn052

182

2838plusmn078

276

503095plusmn043

139

22911plusmn067

231

42993plusmn111

370

33000plusmn074

247

2979plusmn10

3347

250

2627plusmn026

099

826

70plusmn027

102

826

67plusmn042

156

826

55plusmn037

119

2655plusmn037

139

100

2722plusmn068

248

828

02plusmn039

138

82774plusmn034

121

82766plusmn058

169

2766plusmn058

209

502756plusmn053

194

828

26plusmn041

147

828

33plusmn040

141

828

05plusmn056

161

2805plusmn056

200

252787plusmn069

247

829

34plusmn055

189

828

58plusmn041

145

828

59plusmn082

194

2859plusmn082

285

1028

75plusmn078

270

629

44plusmn055

186

529

08plusmn033

114

729

07plusmn060

190

2907plusmn060

207

528

82plusmn015

053

428

71plusmn

021

072

328

88plusmn032

112

428

81plusmn

023

079

2881plusmn

023

079

Theintra-a

ndinterassay

varia

bilityandcoeffi

ciento

fvariatio

nswerec

alculated


=standard

deviationof



100intra-assayCV

=mean

oftheC

Vso

fallruns

lowastAv

erageC


deviation

lowastlowastCV

()=

standard



Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)






RotorGene QMDx

Mx3005P






4 Discussion












5 Conclusion



Abbreviations




Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(c) Continued











Invitrogen
































3 Results








Table2Com

paris

onof

RT-qPC

RSybrGreen

kits(tw

o-ste

p)provided

bythes

amem

anufacturerRe

sults

offived

ifferentq

PCRkitson

arange

ofseventriplicated

ilutio

nsof

EBLV

-1RN

A

Testingof

different

RT-qPC

Rkits(tw

o-ste

p)provided

bythes

amem


Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

IScriptR

T+qP

CRSsoF

ast

QuantiTectR

T+qP

CRQuantitect

Maxim

aRT+qP

CRMaxim

aSS

ViloRT

+qP

CREx

press

qScriptR

T+qP

CRPerfe

cta

AverageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowast

106

5010

51658plusmn013

077

1947plusmn007

034

1650plusmn015

092

1501plusmn

005

035

1609plusmn003

019

1896plusmn004

021

2279plusmn026

115

1747plusmn009

052

1749plusmn010

054

1799plusmn029

159

105

5010

41993plusmn009

045

2289plusmn010

042

1950plusmn006

033

1821plusmn

005

026

1979plusmn003

013

2217plusmn006

025

2594plusmn009

035

2116plusmn029

138

2095plusmn003

015

2127plusmn015

072

104

5010

32328plusmn005

020

2589plusmn006

025

2295plusmn028

123

2168plusmn017

076

2318plusmn016

067

2575plusmn005

018

2960plusmn042

143

2429plusmn029

039

2440plusmn012

049

2455plusmn008

033

103

5010

22661plusmn

021

080

2964plusmn032

107

2643plusmn026

098

2481plusmn

005

019

2694plusmn008

030

2916plusmn023

080

3210plusmn019

060

2778plusmn030

109

2803plusmn074

263

2784plusmn012

042

102

5010

13045plusmn013

042

3220plusmn041

126

3059plusmn045

146

2833plusmn064

225

3140plusmn053

168

3238plusmn061

189

3512plusmn010

028

3046plusmn092

303

3105plusmn038

123

3135plusmn070

222

101

5010

023lowastlowast

13lowastlowast

23lowastlowast

3152plusmn13

3420

13lowastlowast

13lowastlowast

13lowastlowast

3276plusmn070

212

13lowastlowast

23lowastlowast

105

13lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

23lowastlowast

Efficiency

99

99

100

101

90

96

107

97

93

102

1198772

0999

0997

0997

0999

0999

099

9099

1099

7099

4099

8

119910-in

tercept

3665

3955

3631

3473

3767

3938

4183

3801

3850

3801

lowastCV

()=

standard


CVwas

notcalculated

whensomeo

fthe

parallelsfailedto

amplify


num

bero

fpositivesnu

mbero

fexp

erim

ents


erageC


deviation


Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)




















Table3Com

paris

onof

qRT-PC

RSY

BRGreen

(one-step)

kits

Results

offived

ifferentq

RT-PCR

kitson

arange

ofseventriplicated

ilutio

nsof

EBLV

-1in

vitro

RNAtranscrip

tsTestingof

different

qRT-PC

Rkits(one-step)

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Quantitect-1ste

pRo

torG

ene-1step

Verso-1step

SSIIIP

latinium-1ste

pqScript-1

step

AverageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowast

106

2010

61666plusmn008

045

1715plusmn005

029

1699plusmn006

038

1452plusmn019

032

1347plusmn003

019

1779plusmn024

132

1588plusmn022

136

1834plusmn032

172

1622plusmn004

022

1848plusmn004

020

105

2010

52006plusmn010

052

2062plusmn004

017

2040plusmn011

052

1790plusmn021

118

1655plusmn003

019

2113plusmn045

212

1942plusmn020

104

2160plusmn015

071

1964plusmn041

211

2238plusmn015

068

104

2010

42358plusmn014

058

2391plusmn

008

033

2382plusmn020

083

2143plusmn012

057

1986plusmn004

018

2460plusmn020

083

2287plusmn016

069

2517plusmn017

069

2308plusmn027

116

2611plusmn010

038

103

2010

32670plusmn024

091

2745plusmn028

101

2693plusmn003

011

2490plusmn023

092

2313plusmn016

067

2795plusmn026

093

2634plusmn033

124

2863plusmn030

106

2614plusmn010

038

2954plusmn025

086

102

2010

23042plusmn008

026

23lowastlowast

3019plusmn060

198

03lowastlowast

03lowastlowast

3198plusmn017

053

2977plusmn039

132

3174plusmn079

250

2893plusmn008

028

3107plusmn037

118

101

2010

13324plusmn029

087

13lowastlowast

3327plusmn15

3460

03lowastlowast

03lowastlowast

3434plusmn10

3299

3284plusmn044

133

23lowastlowast

2991plusmn

010

033

03lowastlowast

12

03lowastlowast

13lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

23lowastlowast

03lowastlowast

03lowastlowast

Efficiency

98

96

100

94

104

97

94

95

100

87

1198772

0998

0999

0999

0998

0999

099

6099

8099

7099

6099

8

119910-in

tercept

3689

3767

3699

3529

3278

3815

3680

3893

3893

4074

lowastCV

()=

standard


CVwas

notcalculated

whensomeo

fthe

parallelsfailedto

amplify


bero

fpositivesnu

mbero

fexp

erim

ents


erageC


deviation


Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)


Table4Detectio

nlim

itof

PCR(LOD95)for

two-ste

pandon

e-ste

pSY

BRGreen

RT-PCR

Two-ste

pRT

-qPC

RIScriptR

T+qP

CRSsoF

ast

BioR

adQuantiTectR

T+qP

CRQuantitect

QIAGEN

Maxim

aRT+qP

CRMaxim

aFE

RMEN

TAS

SSViloRT

+qP

CREx

press

INVITRO

GEN

qScriptR

T+qP

CRPerfe

cta

QUA

NTA

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

11055

171

8678

3941

2188

4488

44160

8076

3845

2267

34One-stepqR

T-PC

RQuantitect-1ste

pQIAGEN

RotorG

ene-1step

QIAGEN

Verso-1step

FERM

ENTA

SSSIIIP

latinium-1ste

pIN

VITRO

GEN

qScript-1

step

QUA

NTA

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

2040

497

994

127

254

321

641

406

812

2244

1530

101

202

50100

981

1962

lowastAllLO

D95results

werec

arrie

dou

tusin

gProb

itmod

ellowastAllPC

Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)


Table5Analysis

ofreprod

ucibilityof

one-ste

pandtwo-ste

pRT

-PCR

(a)

show

sfor

each

two-ste

pkittheintra-a

ndinterassay

reprod


nsof

EBLV

-1teste

deighttim

esin

each

ofthreeind

ependent

runs


nd(b)sho

wsthe


ucibilityfora

llteste

don

e-ste

pkits

(a)2-ste

pmetho

ds

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CV ()lowastlowast

AverageC

tplusmnSD

CtCV

()lowastlowast

IScriptR

T+qP

CRSsoF

ast

100

3046plusmn041

135

83008plusmn057

190

82982plusmn041

138

83012plusmn046

154

3012plusmn052

174

503111plusmn093

299

63184plusmn10

1319

63080plusmn046

151

83125plusmn080

256

3120plusmn088

283

253249plusmn059

183

63160plusmn067

212

83127plusmn068

219

63179plusmn065

204

3177plusmn080

251

103238plusmn091

281

63269plusmn119

363

63197plusmn076

239

53235plusmn095

294

3237plusmn097

299

53314

mdash1

mdashmdash

03198plusmn005

015

33256plusmn005

015

3227plusmn058

181

05

3217

mdash1

mdashmdash

03211plusmn076

236

33214plusmn076

236

3213plusmn062

193

250

3071plusmn

049

161

830

70plusmn039

128

830

61plusmn

033

108

830

67plusmn041

132

3067plusmn040

129

100

3230plusmn074

228

83203plusmn090

281

83219plusmn040

124

83217plusmn068

211

3217plusmn069

214

5034

02plusmn093

273

63324plusmn048

144

83316plusmn093

287

83347plusmn079

235

3342plusmn085

255

2534

63plusmn10

2295

83378plusmn075

223

634

24plusmn088

258

634

21plusmn

089

259

3425plusmn093

272

103595plusmn11

2312

53526plusmn047

134

534

68plusmn00

63

180

63530plusmn074

209

3526plusmn090

256

53384plusmn062

184

3mdash

mdash0

mdashmdash

034

83plusmn060

171

3384plusmn062

184

QuantiTectR

T+qP

CRQuantitect

250

3137plusmn069

220

83145plusmn052

165

83122plusmn046

147

83135plusmn056

177

3135plusmn055

175

100

3246plusmn080

246

73272plusmn092

281

73390plusmn090

265

73303plusmn087

264

3303plusmn10

5318

503359plusmn060

179

43434plusmn232

675

53366plusmn079

235

63386plusmn12

4363

3387plusmn14

0413

253298plusmn074

224

43358plusmn118

351

53412plusmn207

608

43356plusmn13

3394

3356plusmn13

8410

103354plusmn018

054

33386plusmn061

180

23473plusmn062

179

23404plusmn047

138

3397plusmn065

192

5mdash

mdash0

3552

mdash1

mdashmdash

03552

mdash3552

mdash50

034

09plusmn036

135

836

37plusmn118

326

63297plusmn038

116

834

30plusmn15

419

234

30plusmn15

4448

250

3642plusmn046

643

83366plusmn080

239

83361plusmn

083

246

834

56plusmn19

7376

3456plusmn19

7569

100

3439plusmn234

257

834

60plusmn098

284

834

50plusmn091

265

834

50plusmn089

269

3450plusmn089

258

5036

28plusmn088

475

63507plusmn091

259

734

92plusmn10

4297

73538plusmn13

2344

3538plusmn13

2374

253597plusmn17

2230

536

28plusmn088

242

436

44plusmn078

214

536

23plusmn079

229

3623plusmn079

217

10mdash

mdash0

mdashmdash

03765plusmn079

210

23765plusmn079

210

3765plusmn079

210


(a)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CV ()lowastlowast

AverageC

tplusmnSD

CtCV

()lowastlowast

Maxim

aRT

+qP

CRMaxim

a

100

3183plusmn084

263

3138plusmn022

070

3009plusmn041

137

311plusmn049

157

311plusmn092

295

503335plusmn10

9326

83342plusmn088

264

53112plusmn064

205

83263plusmn087

265

3251plusmn

140

432

253367plusmn088

261

53277plusmn112

340

63247plusmn072

221

73297plusmn090

274

3290plusmn099

302

103716plusmn464

1249

33400plusmn14

1416

43435plusmn13

3388

53517plusmn246

684

3494plusmn263

754

53544plusmn024

068

23865

mdash1

3458plusmn12

4357

33622plusmn074

212

3555plusmn17

6496

25

mdashmdash

03314

mdash1

3347plusmn17

6526

23330plusmn17

6526

3336plusmn12

6377

250

2928plusmn058

199

829

05plusmn038

130

829

04plusmn039

135

829

12plusmn045

155

2912plusmn045

156

100

3053plusmn056

185

830

17plusmn048

159

830

31plusmn

043

143

830

34plusmn050

162

3034plusmn050

164

503154plusmn12

5397

83188plusmn13

7429

83172plusmn056

177

83171plusmn

108

334

3171plusmn

108

340

253300plusmn115

348

63238plusmn079

243

73238plusmn082

254

73256plusmn092

282

3256plusmn092

281

103308plusmn064

193

33302plusmn13

8417

43340plusmn14

6438

43317plusmn115

349

3317plusmn11

5347

503303plusmn015

045

23355

mdash1

3299

mdash1

3315plusmn028

045

3315plusmn028

085

qScriptR

T+qP

CRPerfe

cta

250

2704plusmn024

088

82675plusmn026

098

82677plusmn024

090

82685plusmn025

092

2685plusmn027

101

100

3054plusmn054

176

82984plusmn054

182

83046plusmn10

7350

83028plusmn072

236

3028plusmn079

261

503203plusmn290

905

73072plusmn089

292

83078plusmn082

267

83118plusmn15

4488

3114plusmn17

7568

253250plusmn098

302

73241plusmn

133

410

73363plusmn216

642

53285plusmn14

9452

3276plusmn14

9456

103222plusmn081

252

53287plusmn046

139

33270plusmn056

171

23260plusmn061

187

3251plusmn

069

211

53278plusmn015

045

2mdash

mdash0

3356plusmn15

5461

23318plusmn085

253

3318plusmn10

0303

250

2947plusmn022

074

829

49plusmn054

183

729

82plusmn038

129

829

60plusmn041

129

2960plusmn041

139

100

3099plusmn10

9352

83114plusmn12

6404

83102plusmn052

169

83105plusmn096

308

3105plusmn096

311

503187plusmn060

188

83137plusmn057

180

83313plusmn11

2337

83212plusmn10

8235

3212plusmn10

8335

253335plusmn299

896

73249plusmn068

211

83271plusmn

087

265

83283plusmn17

2457

3283plusmn17

2524

103300plusmn049

147

53345plusmn018

053

23298plusmn074

225

43307plusmn054

142

3307plusmn054

165

53304plusmn10

1306

23303plusmn074

223

23359

mdash1

3314plusmn067

265

3314plusmn067

203


(a)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CV ()lowastlowast

AverageC

tplusmnSD

CtCV

()lowastlowast

SSViloRT

+qP

CREx

press

100

2768plusmn059

213

82782plusmn055

197

82759plusmn029

106

82770plusmn048

172

2770plusmn048

174

502942plusmn062

211

82927plusmn054

185

82921plusmn

060

206

82930plusmn059

201

2930plusmn057

195

253038plusmn067

221

63022plusmn12

4411

82960plusmn048

162

83007plusmn080

264

3004plusmn090

300

103136plusmn049

158

73130plusmn071

227

73192plusmn078

245

63153plusmn066

210

3151plusmn

069

219

53149plusmn053

168

43163plusmn098

308

43164

mdash1

3159plusmn075

238

3157plusmn068

216

05

mdashmdash

0mdash

mdash0

3159

mdash1

3159

mdash3159

mdash100

3178plusmn053

167

830

13plusmn032

107

830

75plusmn065

211

830

89plusmn085

162

3089plusmn085

276

503296plusmn042

129

83134plusmn050

161

83149plusmn097

307

83193plusmn099

199

3193plusmn099

309

253372plusmn10

5310

73215plusmn098

306

83209plusmn022

069

63266plusmn113

228

3266plusmn11

3346

1034

44plusmn060

174

73361plusmn

046

135

53330plusmn043

129

63383plusmn071

146

3383plusmn071

209

53521plusmn

053

151

73264plusmn036

111

53299plusmn006

017

23397plusmn13

5093

3397plusmn13

5398

25

3956plusmn18

6471

334

35

13355plusmn057

169

336

24plusmn332

320

3624plusmn332

916


varia

bilitya

ndcoeffi

ciento

fvariatio

nswerec

alculated


CV=sta



100intra-assayC

V=mean

oftheC

Vso

fallruns

lowastAv

erageC


deviation

lowastlowastCV

()=

standard



Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)

(b)1stepmetho

ds

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CVlowastlowast

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Quantitect1step

502976plusmn064

216

82894plusmn055

189

82927plusmn023

079

82932plusmn047

161

2932plusmn059

106

253090plusmn088

286

82984plusmn10

9365

83098plusmn076

245

83057plusmn091

299

3057plusmn10

3202

103172plusmn10

4326

73205plusmn10

6332

73230plusmn061

188

83203plusmn090

282

3204plusmn090

336

53334plusmn12

0360

63238plusmn074

228

73285plusmn084

255

73286plusmn092

281

3283plusmn096

281

25

3251plusmn

060

185

43390plusmn033

096

43445plusmn15

2442

53362plusmn082

241

3368plusmn12

7377

13338plusmn096

288

43203plusmn026

080

33292plusmn047

143

53277plusmn056

170

3285plusmn079

242

5030

92plusmn047

152

83123plusmn12

9040

83111plusmn13

2041

83108plusmn13

8043

3108plusmn043

138

253177plusmn073

229

83192plusmn14

9048

83191plusmn

093

030

83187plusmn15

7050

3187plusmn051

161

103260plusmn047

144

83281plusmn

061

187

53249plusmn065

199

83263plusmn058

177

3263plusmn057

175

53369plusmn085

253

73382plusmn291

098

53328plusmn250

083

63360plusmn265

089

3357plusmn086

257

25

3436plusmn073

214

53365plusmn18

8063

23327plusmn18

8062

33376plusmn19

6066

3380plusmn080

236

134

55plusmn17

7513

334

97plusmn117

041

53392plusmn341

116

134

48plusmn324

111

3445plusmn13

7397


(b)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CVlowastlowast

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

RotorG

ene1

step

1000

2640plusmn045

169

82374plusmn040

167

82701plusmn

118

438

82572plusmn16

2258

2572plusmn16

2632

500

2749plusmn046

166

82429plusmn034

139

82742plusmn10

4378

82640plusmn16

6228

2640plusmn16

6628

250

2873plusmn076

265

82490plusmn049

196

82927plusmn060

204

72756plusmn209

222

2756plusmn209

758

100

3070plusmn021

067

32550

mdash1

3119plusmn066

211

23000plusmn224

139

3000plusmn224

746

503031

mdash1

mdashmdash

0mdash

mdash0

3031

mdash3031

mdash25

3112plusmn050

161

2mdash

mdash0

mdashmdash

03212plusmn050

161

3212plusmn050

161

5028

87plusmn099

341

829

25plusmn074

252

82797plusmn065

233

828

70plusmn079

276

2870plusmn094

329

2529

49plusmn10

1344

830

36plusmn070

229

828

57plusmn039

136

829

47plusmn070

236

2947plusmn10

3350

1030

29plusmn12

0398

830

61plusmn

144

470

828

76plusmn040

139

829

89plusmn10

1336

2989plusmn13

4450

530

82plusmn10

6343

63147plusmn13

0412

629

90plusmn040

135

830

73plusmn092

297

3065plusmn11

2366

25

3087plusmn114

368

63169plusmn057

181

529

10plusmn037

126

530

55plusmn069

225

3057plusmn13

1429

13122

mdash1

3153

mdash1

3201plusmn

044

137

53159plusmn044

137

3183plusmn048

151

Verso1step

500

2650plusmn018

068

82656plusmn014

053

82679plusmn023

085

82661plusmn

018

069

2661plusmn

022

082

250

2743plusmn018

065

82796plusmn027

098

82789plusmn036

127

82776plusmn027

097

2776plusmn036

130

100

2894plusmn050

172

82942plusmn053

180

82991plusmn

118

396

82943plusmn074

249

2943plusmn087

295

503049plusmn044

143

73138plusmn086

274

83129plusmn15

2487

83105plusmn094

301

3108plusmn10

9350

253282plusmn114

346

73290plusmn052

157

53250plusmn092

282

73274plusmn086

262

3272plusmn090

274

103342plusmn097

291

33522plusmn10

5299

23442

mdash1

3435plusmn10

1295

3419plusmn118

344

250

2939plusmn048

164

829

40plusmn023

079

829

10plusmn053

183

829

20plusmn042

142

2930plusmn044

150

100

3055plusmn025

080

830

32plusmn038

125

830

02plusmn024

082

630

29plusmn029

096

3032plusmn036

119

503116plusmn050

159

83110plusmn052

168

830

87plusmn042

135

73104plusmn048

154

3105plusmn048

153

253171plusmn

032

102

73161plusmn

042

134

83135plusmn058

184

83156plusmn044

140

3155plusmn046

147

103186plusmn054

169

73176plusmn029

091

73165plusmn064

201

43176plusmn049

154

3177plusmn046

145

53309plusmn098

297

53313

13343plusmn074

223

43322plusmn086

260

3326plusmn080

239


(b)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CVlowastlowast

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

qScript1

step

1000

2324plusmn012

053

82297plusmn024

103

82321plusmn

009

037

82314plusmn015

064

2314plusmn020

086

750

2364plusmn021

088

82329plusmn008

035

82352plusmn028

121

82348plusmn019

081

2348plusmn025

106

500

2446plusmn032

132

82406plusmn024

098

82407plusmn029

122

82419plusmn028

117

2419plusmn033

137

250

2538plusmn027

107

82493plusmn026

105

82514plusmn034

137

82515plusmn029

116

2515plusmn034

135

100

2626plusmn031

116

82579plusmn024

093

72595plusmn047

180

52600plusmn034

130

2602plusmn038

146

50mdash

mdash0

mdashmdash

02645plusmn018

070

22645plusmn018

070

2645plusmn018

070

2500

2813plusmn083

294

82778plusmn045

160

826

32plusmn030

113

82741plusmn

052

189

2741plusmn

097

353

1000

2878plusmn009

033

829

40plusmn15

8539

824

89plusmn030

122

82769plusmn066

231

2769plusmn223

805

750

2908plusmn024

082

828

92plusmn025

085

828

75plusmn011

038

828

92plusmn020

068

2892plusmn024

084

500

3069plusmn048

157

729

76plusmn055

186

82763plusmn056

201

529

36plusmn053

181

2966plusmn12

6424

250

3122plusmn023

073

730

05plusmn037

124

8mdash

mdash0

3064plusmn030

099

3060plusmn067

220

100

mdashmdash

030

00

mdash1

mdashmdash

030

00

mdash30

00

mdash

SSIIIP

latinium

1step

1000

2479plusmn024

097

82381plusmn

021

089

82446plusmn015

060

82435plusmn020

082

2435plusmn046

188

750

2518plusmn024

095

82430plusmn012

051

82497plusmn010

042

82482plusmn016

063

2482plusmn041

167

500

2666plusmn082

307

82536plusmn023

092

82619plusmn024

092

82607plusmn043

164

2607plusmn073

282

250

2866plusmn086

301

82838plusmn19

1672

82897plusmn059

205

82867plusmn112

393

2867plusmn12

2427

100

2917plusmn053

180

82778plusmn049

175

82816plusmn053

189

72837plusmn052

182

2838plusmn078

276

503095plusmn043

139

22911plusmn067

231

42993plusmn111

370

33000plusmn074

247

2979plusmn10

3347

250

2627plusmn026

099

826

70plusmn027

102

826

67plusmn042

156

826

55plusmn037

119

2655plusmn037

139

100

2722plusmn068

248

828

02plusmn039

138

82774plusmn034

121

82766plusmn058

169

2766plusmn058

209

502756plusmn053

194

828

26plusmn041

147

828

33plusmn040

141

828

05plusmn056

161

2805plusmn056

200

252787plusmn069

247

829

34plusmn055

189

828

58plusmn041

145

828

59plusmn082

194

2859plusmn082

285

1028

75plusmn078

270

629

44plusmn055

186

529

08plusmn033

114

729

07plusmn060

190

2907plusmn060

207

528

82plusmn015

053

428

71plusmn

021

072

328

88plusmn032

112

428

81plusmn

023

079

2881plusmn

023

079

Theintra-a

ndinterassay

varia

bilityandcoeffi

ciento

fvariatio

nswerec

alculated


=standard

deviationof



100intra-assayCV

=mean

oftheC

Vso

fallruns

lowastAv

erageC


deviation

lowastlowastCV

()=

standard



Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)






RotorGene QMDx

Mx3005P






4 Discussion












5 Conclusion



Abbreviations




Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology













3 Results








Table2Com

paris

onof

RT-qPC

RSybrGreen

kits(tw

o-ste

p)provided

bythes

amem

anufacturerRe

sults

offived

ifferentq

PCRkitson

arange

ofseventriplicated

ilutio

nsof

EBLV

-1RN

A

Testingof

different

RT-qPC

Rkits(tw

o-ste

p)provided

bythes

amem


Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

IScriptR

T+qP

CRSsoF

ast

QuantiTectR

T+qP

CRQuantitect

Maxim

aRT+qP

CRMaxim

aSS

ViloRT

+qP

CREx

press

qScriptR

T+qP

CRPerfe

cta

AverageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowast

106

5010

51658plusmn013

077

1947plusmn007

034

1650plusmn015

092

1501plusmn

005

035

1609plusmn003

019

1896plusmn004

021

2279plusmn026

115

1747plusmn009

052

1749plusmn010

054

1799plusmn029

159

105

5010

41993plusmn009

045

2289plusmn010

042

1950plusmn006

033

1821plusmn

005

026

1979plusmn003

013

2217plusmn006

025

2594plusmn009

035

2116plusmn029

138

2095plusmn003

015

2127plusmn015

072

104

5010

32328plusmn005

020

2589plusmn006

025

2295plusmn028

123

2168plusmn017

076

2318plusmn016

067

2575plusmn005

018

2960plusmn042

143

2429plusmn029

039

2440plusmn012

049

2455plusmn008

033

103

5010

22661plusmn

021

080

2964plusmn032

107

2643plusmn026

098

2481plusmn

005

019

2694plusmn008

030

2916plusmn023

080

3210plusmn019

060

2778plusmn030

109

2803plusmn074

263

2784plusmn012

042

102

5010

13045plusmn013

042

3220plusmn041

126

3059plusmn045

146

2833plusmn064

225

3140plusmn053

168

3238plusmn061

189

3512plusmn010

028

3046plusmn092

303

3105plusmn038

123

3135plusmn070

222

101

5010

023lowastlowast

13lowastlowast

23lowastlowast

3152plusmn13

3420

13lowastlowast

13lowastlowast

13lowastlowast

3276plusmn070

212

13lowastlowast

23lowastlowast

105

13lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

23lowastlowast

Efficiency

99

99

100

101

90

96

107

97

93

102

1198772

0999

0997

0997

0999

0999

099

9099

1099

7099

4099

8

119910-in

tercept

3665

3955

3631

3473

3767

3938

4183

3801

3850

3801

lowastCV

()=

standard


CVwas

notcalculated

whensomeo

fthe

parallelsfailedto

amplify


num

bero

fpositivesnu

mbero

fexp

erim

ents


erageC


deviation


Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)




















Table3Com

paris

onof

qRT-PC

RSY

BRGreen

(one-step)

kits

Results

offived

ifferentq

RT-PCR

kitson

arange

ofseventriplicated

ilutio

nsof

EBLV

-1in

vitro

RNAtranscrip

tsTestingof

different

qRT-PC

Rkits(one-step)

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Quantitect-1ste

pRo

torG

ene-1step

Verso-1step

SSIIIP

latinium-1ste

pqScript-1

step

AverageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowast

106

2010

61666plusmn008

045

1715plusmn005

029

1699plusmn006

038

1452plusmn019

032

1347plusmn003

019

1779plusmn024

132

1588plusmn022

136

1834plusmn032

172

1622plusmn004

022

1848plusmn004

020

105

2010

52006plusmn010

052

2062plusmn004

017

2040plusmn011

052

1790plusmn021

118

1655plusmn003

019

2113plusmn045

212

1942plusmn020

104

2160plusmn015

071

1964plusmn041

211

2238plusmn015

068

104

2010

42358plusmn014

058

2391plusmn

008

033

2382plusmn020

083

2143plusmn012

057

1986plusmn004

018

2460plusmn020

083

2287plusmn016

069

2517plusmn017

069

2308plusmn027

116

2611plusmn010

038

103

2010

32670plusmn024

091

2745plusmn028

101

2693plusmn003

011

2490plusmn023

092

2313plusmn016

067

2795plusmn026

093

2634plusmn033

124

2863plusmn030

106

2614plusmn010

038

2954plusmn025

086

102

2010

23042plusmn008

026

23lowastlowast

3019plusmn060

198

03lowastlowast

03lowastlowast

3198plusmn017

053

2977plusmn039

132

3174plusmn079

250

2893plusmn008

028

3107plusmn037

118

101

2010

13324plusmn029

087

13lowastlowast

3327plusmn15

3460

03lowastlowast

03lowastlowast

3434plusmn10

3299

3284plusmn044

133

23lowastlowast

2991plusmn

010

033

03lowastlowast

12

03lowastlowast

13lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

23lowastlowast

03lowastlowast

03lowastlowast

Efficiency

98

96

100

94

104

97

94

95

100

87

1198772

0998

0999

0999

0998

0999

099

6099

8099

7099

6099

8

119910-in

tercept

3689

3767

3699

3529

3278

3815

3680

3893

3893

4074

lowastCV

()=

standard


CVwas

notcalculated

whensomeo

fthe

parallelsfailedto

amplify


bero

fpositivesnu

mbero

fexp

erim

ents


erageC


deviation


Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)


Table4Detectio

nlim

itof

PCR(LOD95)for

two-ste

pandon

e-ste

pSY

BRGreen

RT-PCR

Two-ste

pRT

-qPC

RIScriptR

T+qP

CRSsoF

ast

BioR

adQuantiTectR

T+qP

CRQuantitect

QIAGEN

Maxim

aRT+qP

CRMaxim

aFE

RMEN

TAS

SSViloRT

+qP

CREx

press

INVITRO

GEN

qScriptR

T+qP

CRPerfe

cta

QUA

NTA

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

11055

171

8678

3941

2188

4488

44160

8076

3845

2267

34One-stepqR

T-PC

RQuantitect-1ste

pQIAGEN

RotorG

ene-1step

QIAGEN

Verso-1step

FERM

ENTA

SSSIIIP

latinium-1ste

pIN

VITRO

GEN

qScript-1

step

QUA

NTA

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

2040

497

994

127

254

321

641

406

812

2244

1530

101

202

50100

981

1962

lowastAllLO

D95results

werec

arrie

dou

tusin

gProb

itmod

ellowastAllPC

Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)


Table5Analysis

ofreprod

ucibilityof

one-ste

pandtwo-ste

pRT

-PCR

(a)

show

sfor

each

two-ste

pkittheintra-a

ndinterassay

reprod


nsof

EBLV

-1teste

deighttim

esin

each

ofthreeind

ependent

runs


nd(b)sho

wsthe


ucibilityfora

llteste

don

e-ste

pkits

(a)2-ste

pmetho

ds

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CV ()lowastlowast

AverageC

tplusmnSD

CtCV

()lowastlowast

IScriptR

T+qP

CRSsoF

ast

100

3046plusmn041

135

83008plusmn057

190

82982plusmn041

138

83012plusmn046

154

3012plusmn052

174

503111plusmn093

299

63184plusmn10

1319

63080plusmn046

151

83125plusmn080

256

3120plusmn088

283

253249plusmn059

183

63160plusmn067

212

83127plusmn068

219

63179plusmn065

204

3177plusmn080

251

103238plusmn091

281

63269plusmn119

363

63197plusmn076

239

53235plusmn095

294

3237plusmn097

299

53314

mdash1

mdashmdash

03198plusmn005

015

33256plusmn005

015

3227plusmn058

181

05

3217

mdash1

mdashmdash

03211plusmn076

236

33214plusmn076

236

3213plusmn062

193

250

3071plusmn

049

161

830

70plusmn039

128

830

61plusmn

033

108

830

67plusmn041

132

3067plusmn040

129

100

3230plusmn074

228

83203plusmn090

281

83219plusmn040

124

83217plusmn068

211

3217plusmn069

214

5034

02plusmn093

273

63324plusmn048

144

83316plusmn093

287

83347plusmn079

235

3342plusmn085

255

2534

63plusmn10

2295

83378plusmn075

223

634

24plusmn088

258

634

21plusmn

089

259

3425plusmn093

272

103595plusmn11

2312

53526plusmn047

134

534

68plusmn00

63

180

63530plusmn074

209

3526plusmn090

256

53384plusmn062

184

3mdash

mdash0

mdashmdash

034

83plusmn060

171

3384plusmn062

184

QuantiTectR

T+qP

CRQuantitect

250

3137plusmn069

220

83145plusmn052

165

83122plusmn046

147

83135plusmn056

177

3135plusmn055

175

100

3246plusmn080

246

73272plusmn092

281

73390plusmn090

265

73303plusmn087

264

3303plusmn10

5318

503359plusmn060

179

43434plusmn232

675

53366plusmn079

235

63386plusmn12

4363

3387plusmn14

0413

253298plusmn074

224

43358plusmn118

351

53412plusmn207

608

43356plusmn13

3394

3356plusmn13

8410

103354plusmn018

054

33386plusmn061

180

23473plusmn062

179

23404plusmn047

138

3397plusmn065

192

5mdash

mdash0

3552

mdash1

mdashmdash

03552

mdash3552

mdash50

034

09plusmn036

135

836

37plusmn118

326

63297plusmn038

116

834

30plusmn15

419

234

30plusmn15

4448

250

3642plusmn046

643

83366plusmn080

239

83361plusmn

083

246

834

56plusmn19

7376

3456plusmn19

7569

100

3439plusmn234

257

834

60plusmn098

284

834

50plusmn091

265

834

50plusmn089

269

3450plusmn089

258

5036

28plusmn088

475

63507plusmn091

259

734

92plusmn10

4297

73538plusmn13

2344

3538plusmn13

2374

253597plusmn17

2230

536

28plusmn088

242

436

44plusmn078

214

536

23plusmn079

229

3623plusmn079

217

10mdash

mdash0

mdashmdash

03765plusmn079

210

23765plusmn079

210

3765plusmn079

210


(a)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CV ()lowastlowast

AverageC

tplusmnSD

CtCV

()lowastlowast

Maxim

aRT

+qP

CRMaxim

a

100

3183plusmn084

263

3138plusmn022

070

3009plusmn041

137

311plusmn049

157

311plusmn092

295

503335plusmn10

9326

83342plusmn088

264

53112plusmn064

205

83263plusmn087

265

3251plusmn

140

432

253367plusmn088

261

53277plusmn112

340

63247plusmn072

221

73297plusmn090

274

3290plusmn099

302

103716plusmn464

1249

33400plusmn14

1416

43435plusmn13

3388

53517plusmn246

684

3494plusmn263

754

53544plusmn024

068

23865

mdash1

3458plusmn12

4357

33622plusmn074

212

3555plusmn17

6496

25

mdashmdash

03314

mdash1

3347plusmn17

6526

23330plusmn17

6526

3336plusmn12

6377

250

2928plusmn058

199

829

05plusmn038

130

829

04plusmn039

135

829

12plusmn045

155

2912plusmn045

156

100

3053plusmn056

185

830

17plusmn048

159

830

31plusmn

043

143

830

34plusmn050

162

3034plusmn050

164

503154plusmn12

5397

83188plusmn13

7429

83172plusmn056

177

83171plusmn

108

334

3171plusmn

108

340

253300plusmn115

348

63238plusmn079

243

73238plusmn082

254

73256plusmn092

282

3256plusmn092

281

103308plusmn064

193

33302plusmn13

8417

43340plusmn14

6438

43317plusmn115

349

3317plusmn11

5347

503303plusmn015

045

23355

mdash1

3299

mdash1

3315plusmn028

045

3315plusmn028

085

qScriptR

T+qP

CRPerfe

cta

250

2704plusmn024

088

82675plusmn026

098

82677plusmn024

090

82685plusmn025

092

2685plusmn027

101

100

3054plusmn054

176

82984plusmn054

182

83046plusmn10

7350

83028plusmn072

236

3028plusmn079

261

503203plusmn290

905

73072plusmn089

292

83078plusmn082

267

83118plusmn15

4488

3114plusmn17

7568

253250plusmn098

302

73241plusmn

133

410

73363plusmn216

642

53285plusmn14

9452

3276plusmn14

9456

103222plusmn081

252

53287plusmn046

139

33270plusmn056

171

23260plusmn061

187

3251plusmn

069

211

53278plusmn015

045

2mdash

mdash0

3356plusmn15

5461

23318plusmn085

253

3318plusmn10

0303

250

2947plusmn022

074

829

49plusmn054

183

729

82plusmn038

129

829

60plusmn041

129

2960plusmn041

139

100

3099plusmn10

9352

83114plusmn12

6404

83102plusmn052

169

83105plusmn096

308

3105plusmn096

311

503187plusmn060

188

83137plusmn057

180

83313plusmn11

2337

83212plusmn10

8235

3212plusmn10

8335

253335plusmn299

896

73249plusmn068

211

83271plusmn

087

265

83283plusmn17

2457

3283plusmn17

2524

103300plusmn049

147

53345plusmn018

053

23298plusmn074

225

43307plusmn054

142

3307plusmn054

165

53304plusmn10

1306

23303plusmn074

223

23359

mdash1

3314plusmn067

265

3314plusmn067

203


(a)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CV ()lowastlowast

AverageC

tplusmnSD

CtCV

()lowastlowast

SSViloRT

+qP

CREx

press

100

2768plusmn059

213

82782plusmn055

197

82759plusmn029

106

82770plusmn048

172

2770plusmn048

174

502942plusmn062

211

82927plusmn054

185

82921plusmn

060

206

82930plusmn059

201

2930plusmn057

195

253038plusmn067

221

63022plusmn12

4411

82960plusmn048

162

83007plusmn080

264

3004plusmn090

300

103136plusmn049

158

73130plusmn071

227

73192plusmn078

245

63153plusmn066

210

3151plusmn

069

219

53149plusmn053

168

43163plusmn098

308

43164

mdash1

3159plusmn075

238

3157plusmn068

216

05

mdashmdash

0mdash

mdash0

3159

mdash1

3159

mdash3159

mdash100

3178plusmn053

167

830

13plusmn032

107

830

75plusmn065

211

830

89plusmn085

162

3089plusmn085

276

503296plusmn042

129

83134plusmn050

161

83149plusmn097

307

83193plusmn099

199

3193plusmn099

309

253372plusmn10

5310

73215plusmn098

306

83209plusmn022

069

63266plusmn113

228

3266plusmn11

3346

1034

44plusmn060

174

73361plusmn

046

135

53330plusmn043

129

63383plusmn071

146

3383plusmn071

209

53521plusmn

053

151

73264plusmn036

111

53299plusmn006

017

23397plusmn13

5093

3397plusmn13

5398

25

3956plusmn18

6471

334

35

13355plusmn057

169

336

24plusmn332

320

3624plusmn332

916


varia

bilitya

ndcoeffi

ciento

fvariatio

nswerec

alculated


CV=sta



100intra-assayC

V=mean

oftheC

Vso

fallruns

lowastAv

erageC


deviation

lowastlowastCV

()=

standard



Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)

(b)1stepmetho

ds

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CVlowastlowast

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Quantitect1step

502976plusmn064

216

82894plusmn055

189

82927plusmn023

079

82932plusmn047

161

2932plusmn059

106

253090plusmn088

286

82984plusmn10

9365

83098plusmn076

245

83057plusmn091

299

3057plusmn10

3202

103172plusmn10

4326

73205plusmn10

6332

73230plusmn061

188

83203plusmn090

282

3204plusmn090

336

53334plusmn12

0360

63238plusmn074

228

73285plusmn084

255

73286plusmn092

281

3283plusmn096

281

25

3251plusmn

060

185

43390plusmn033

096

43445plusmn15

2442

53362plusmn082

241

3368plusmn12

7377

13338plusmn096

288

43203plusmn026

080

33292plusmn047

143

53277plusmn056

170

3285plusmn079

242

5030

92plusmn047

152

83123plusmn12

9040

83111plusmn13

2041

83108plusmn13

8043

3108plusmn043

138

253177plusmn073

229

83192plusmn14

9048

83191plusmn

093

030

83187plusmn15

7050

3187plusmn051

161

103260plusmn047

144

83281plusmn

061

187

53249plusmn065

199

83263plusmn058

177

3263plusmn057

175

53369plusmn085

253

73382plusmn291

098

53328plusmn250

083

63360plusmn265

089

3357plusmn086

257

25

3436plusmn073

214

53365plusmn18

8063

23327plusmn18

8062

33376plusmn19

6066

3380plusmn080

236

134

55plusmn17

7513

334

97plusmn117

041

53392plusmn341

116

134

48plusmn324

111

3445plusmn13

7397


(b)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CVlowastlowast

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

RotorG

ene1

step

1000

2640plusmn045

169

82374plusmn040

167

82701plusmn

118

438

82572plusmn16

2258

2572plusmn16

2632

500

2749plusmn046

166

82429plusmn034

139

82742plusmn10

4378

82640plusmn16

6228

2640plusmn16

6628

250

2873plusmn076

265

82490plusmn049

196

82927plusmn060

204

72756plusmn209

222

2756plusmn209

758

100

3070plusmn021

067

32550

mdash1

3119plusmn066

211

23000plusmn224

139

3000plusmn224

746

503031

mdash1

mdashmdash

0mdash

mdash0

3031

mdash3031

mdash25

3112plusmn050

161

2mdash

mdash0

mdashmdash

03212plusmn050

161

3212plusmn050

161

5028

87plusmn099

341

829

25plusmn074

252

82797plusmn065

233

828

70plusmn079

276

2870plusmn094

329

2529

49plusmn10

1344

830

36plusmn070

229

828

57plusmn039

136

829

47plusmn070

236

2947plusmn10

3350

1030

29plusmn12

0398

830

61plusmn

144

470

828

76plusmn040

139

829

89plusmn10

1336

2989plusmn13

4450

530

82plusmn10

6343

63147plusmn13

0412

629

90plusmn040

135

830

73plusmn092

297

3065plusmn11

2366

25

3087plusmn114

368

63169plusmn057

181

529

10plusmn037

126

530

55plusmn069

225

3057plusmn13

1429

13122

mdash1

3153

mdash1

3201plusmn

044

137

53159plusmn044

137

3183plusmn048

151

Verso1step

500

2650plusmn018

068

82656plusmn014

053

82679plusmn023

085

82661plusmn

018

069

2661plusmn

022

082

250

2743plusmn018

065

82796plusmn027

098

82789plusmn036

127

82776plusmn027

097

2776plusmn036

130

100

2894plusmn050

172

82942plusmn053

180

82991plusmn

118

396

82943plusmn074

249

2943plusmn087

295

503049plusmn044

143

73138plusmn086

274

83129plusmn15

2487

83105plusmn094

301

3108plusmn10

9350

253282plusmn114

346

73290plusmn052

157

53250plusmn092

282

73274plusmn086

262

3272plusmn090

274

103342plusmn097

291

33522plusmn10

5299

23442

mdash1

3435plusmn10

1295

3419plusmn118

344

250

2939plusmn048

164

829

40plusmn023

079

829

10plusmn053

183

829

20plusmn042

142

2930plusmn044

150

100

3055plusmn025

080

830

32plusmn038

125

830

02plusmn024

082

630

29plusmn029

096

3032plusmn036

119

503116plusmn050

159

83110plusmn052

168

830

87plusmn042

135

73104plusmn048

154

3105plusmn048

153

253171plusmn

032

102

73161plusmn

042

134

83135plusmn058

184

83156plusmn044

140

3155plusmn046

147

103186plusmn054

169

73176plusmn029

091

73165plusmn064

201

43176plusmn049

154

3177plusmn046

145

53309plusmn098

297

53313

13343plusmn074

223

43322plusmn086

260

3326plusmn080

239


(b)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CVlowastlowast

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

qScript1

step

1000

2324plusmn012

053

82297plusmn024

103

82321plusmn

009

037

82314plusmn015

064

2314plusmn020

086

750

2364plusmn021

088

82329plusmn008

035

82352plusmn028

121

82348plusmn019

081

2348plusmn025

106

500

2446plusmn032

132

82406plusmn024

098

82407plusmn029

122

82419plusmn028

117

2419plusmn033

137

250

2538plusmn027

107

82493plusmn026

105

82514plusmn034

137

82515plusmn029

116

2515plusmn034

135

100

2626plusmn031

116

82579plusmn024

093

72595plusmn047

180

52600plusmn034

130

2602plusmn038

146

50mdash

mdash0

mdashmdash

02645plusmn018

070

22645plusmn018

070

2645plusmn018

070

2500

2813plusmn083

294

82778plusmn045

160

826

32plusmn030

113

82741plusmn

052

189

2741plusmn

097

353

1000

2878plusmn009

033

829

40plusmn15

8539

824

89plusmn030

122

82769plusmn066

231

2769plusmn223

805

750

2908plusmn024

082

828

92plusmn025

085

828

75plusmn011

038

828

92plusmn020

068

2892plusmn024

084

500

3069plusmn048

157

729

76plusmn055

186

82763plusmn056

201

529

36plusmn053

181

2966plusmn12

6424

250

3122plusmn023

073

730

05plusmn037

124

8mdash

mdash0

3064plusmn030

099

3060plusmn067

220

100

mdashmdash

030

00

mdash1

mdashmdash

030

00

mdash30

00

mdash

SSIIIP

latinium

1step

1000

2479plusmn024

097

82381plusmn

021

089

82446plusmn015

060

82435plusmn020

082

2435plusmn046

188

750

2518plusmn024

095

82430plusmn012

051

82497plusmn010

042

82482plusmn016

063

2482plusmn041

167

500

2666plusmn082

307

82536plusmn023

092

82619plusmn024

092

82607plusmn043

164

2607plusmn073

282

250

2866plusmn086

301

82838plusmn19

1672

82897plusmn059

205

82867plusmn112

393

2867plusmn12

2427

100

2917plusmn053

180

82778plusmn049

175

82816plusmn053

189

72837plusmn052

182

2838plusmn078

276

503095plusmn043

139

22911plusmn067

231

42993plusmn111

370

33000plusmn074

247

2979plusmn10

3347

250

2627plusmn026

099

826

70plusmn027

102

826

67plusmn042

156

826

55plusmn037

119

2655plusmn037

139

100

2722plusmn068

248

828

02plusmn039

138

82774plusmn034

121

82766plusmn058

169

2766plusmn058

209

502756plusmn053

194

828

26plusmn041

147

828

33plusmn040

141

828

05plusmn056

161

2805plusmn056

200

252787plusmn069

247

829

34plusmn055

189

828

58plusmn041

145

828

59plusmn082

194

2859plusmn082

285

1028

75plusmn078

270

629

44plusmn055

186

529

08plusmn033

114

729

07plusmn060

190

2907plusmn060

207

528

82plusmn015

053

428

71plusmn

021

072

328

88plusmn032

112

428

81plusmn

023

079

2881plusmn

023

079

Theintra-a

ndinterassay

varia

bilityandcoeffi

ciento

fvariatio

nswerec

alculated


=standard

deviationof



100intra-assayCV

=mean

oftheC

Vso

fallruns

lowastAv

erageC


deviation

lowastlowastCV

()=

standard



Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)






RotorGene QMDx

Mx3005P






4 Discussion












5 Conclusion



Abbreviations




Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table2Com

paris

onof

RT-qPC

RSybrGreen

kits(tw

o-ste

p)provided

bythes

amem

anufacturerRe

sults

offived

ifferentq

PCRkitson

arange

ofseventriplicated

ilutio

nsof

EBLV

-1RN

A

Testingof

different

RT-qPC

Rkits(tw

o-ste

p)provided

bythes

amem


Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

IScriptR

T+qP

CRSsoF

ast

QuantiTectR

T+qP

CRQuantitect

Maxim

aRT+qP

CRMaxim

aSS

ViloRT

+qP

CREx

press

qScriptR

T+qP

CRPerfe

cta

AverageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowast

106

5010

51658plusmn013

077

1947plusmn007

034

1650plusmn015

092

1501plusmn

005

035

1609plusmn003

019

1896plusmn004

021

2279plusmn026

115

1747plusmn009

052

1749plusmn010

054

1799plusmn029

159

105

5010

41993plusmn009

045

2289plusmn010

042

1950plusmn006

033

1821plusmn

005

026

1979plusmn003

013

2217plusmn006

025

2594plusmn009

035

2116plusmn029

138

2095plusmn003

015

2127plusmn015

072

104

5010

32328plusmn005

020

2589plusmn006

025

2295plusmn028

123

2168plusmn017

076

2318plusmn016

067

2575plusmn005

018

2960plusmn042

143

2429plusmn029

039

2440plusmn012

049

2455plusmn008

033

103

5010

22661plusmn

021

080

2964plusmn032

107

2643plusmn026

098

2481plusmn

005

019

2694plusmn008

030

2916plusmn023

080

3210plusmn019

060

2778plusmn030

109

2803plusmn074

263

2784plusmn012

042

102

5010

13045plusmn013

042

3220plusmn041

126

3059plusmn045

146

2833plusmn064

225

3140plusmn053

168

3238plusmn061

189

3512plusmn010

028

3046plusmn092

303

3105plusmn038

123

3135plusmn070

222

101

5010

023lowastlowast

13lowastlowast

23lowastlowast

3152plusmn13

3420

13lowastlowast

13lowastlowast

13lowastlowast

3276plusmn070

212

13lowastlowast

23lowastlowast

105

13lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

23lowastlowast

Efficiency

99

99

100

101

90

96

107

97

93

102

1198772

0999

0997

0997

0999

0999

099

9099

1099

7099

4099

8

119910-in

tercept

3665

3955

3631

3473

3767

3938

4183

3801

3850

3801

lowastCV

()=

standard


CVwas

notcalculated

whensomeo

fthe

parallelsfailedto

amplify


num

bero

fpositivesnu

mbero

fexp

erim

ents


erageC


deviation


Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)




















Table3Com

paris

onof

qRT-PC

RSY

BRGreen

(one-step)

kits

Results

offived

ifferentq

RT-PCR

kitson

arange

ofseventriplicated

ilutio

nsof

EBLV

-1in

vitro

RNAtranscrip

tsTestingof

different

qRT-PC

Rkits(one-step)

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Quantitect-1ste

pRo

torG

ene-1step

Verso-1step

SSIIIP

latinium-1ste

pqScript-1

step

AverageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowast

106

2010

61666plusmn008

045

1715plusmn005

029

1699plusmn006

038

1452plusmn019

032

1347plusmn003

019

1779plusmn024

132

1588plusmn022

136

1834plusmn032

172

1622plusmn004

022

1848plusmn004

020

105

2010

52006plusmn010

052

2062plusmn004

017

2040plusmn011

052

1790plusmn021

118

1655plusmn003

019

2113plusmn045

212

1942plusmn020

104

2160plusmn015

071

1964plusmn041

211

2238plusmn015

068

104

2010

42358plusmn014

058

2391plusmn

008

033

2382plusmn020

083

2143plusmn012

057

1986plusmn004

018

2460plusmn020

083

2287plusmn016

069

2517plusmn017

069

2308plusmn027

116

2611plusmn010

038

103

2010

32670plusmn024

091

2745plusmn028

101

2693plusmn003

011

2490plusmn023

092

2313plusmn016

067

2795plusmn026

093

2634plusmn033

124

2863plusmn030

106

2614plusmn010

038

2954plusmn025

086

102

2010

23042plusmn008

026

23lowastlowast

3019plusmn060

198

03lowastlowast

03lowastlowast

3198plusmn017

053

2977plusmn039

132

3174plusmn079

250

2893plusmn008

028

3107plusmn037

118

101

2010

13324plusmn029

087

13lowastlowast

3327plusmn15

3460

03lowastlowast

03lowastlowast

3434plusmn10

3299

3284plusmn044

133

23lowastlowast

2991plusmn

010

033

03lowastlowast

12

03lowastlowast

13lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

23lowastlowast

03lowastlowast

03lowastlowast

Efficiency

98

96

100

94

104

97

94

95

100

87

1198772

0998

0999

0999

0998

0999

099

6099

8099

7099

6099

8

119910-in

tercept

3689

3767

3699

3529

3278

3815

3680

3893

3893

4074

lowastCV

()=

standard


CVwas

notcalculated

whensomeo

fthe

parallelsfailedto

amplify


bero

fpositivesnu

mbero

fexp

erim

ents


erageC


deviation


Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)


Table4Detectio

nlim

itof

PCR(LOD95)for

two-ste

pandon

e-ste

pSY

BRGreen

RT-PCR

Two-ste

pRT

-qPC

RIScriptR

T+qP

CRSsoF

ast

BioR

adQuantiTectR

T+qP

CRQuantitect

QIAGEN

Maxim

aRT+qP

CRMaxim

aFE

RMEN

TAS

SSViloRT

+qP

CREx

press

INVITRO

GEN

qScriptR

T+qP

CRPerfe

cta

QUA

NTA

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

11055

171

8678

3941

2188

4488

44160

8076

3845

2267

34One-stepqR

T-PC

RQuantitect-1ste

pQIAGEN

RotorG

ene-1step

QIAGEN

Verso-1step

FERM

ENTA

SSSIIIP

latinium-1ste

pIN

VITRO

GEN

qScript-1

step

QUA

NTA

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

2040

497

994

127

254

321

641

406

812

2244

1530

101

202

50100

981

1962

lowastAllLO

D95results

werec

arrie

dou

tusin

gProb

itmod

ellowastAllPC

Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)


Table5Analysis

ofreprod

ucibilityof

one-ste

pandtwo-ste

pRT

-PCR

(a)

show

sfor

each

two-ste

pkittheintra-a

ndinterassay

reprod


nsof

EBLV

-1teste

deighttim

esin

each

ofthreeind

ependent

runs


nd(b)sho

wsthe


ucibilityfora

llteste

don

e-ste

pkits

(a)2-ste

pmetho

ds

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CV ()lowastlowast

AverageC

tplusmnSD

CtCV

()lowastlowast

IScriptR

T+qP

CRSsoF

ast

100

3046plusmn041

135

83008plusmn057

190

82982plusmn041

138

83012plusmn046

154

3012plusmn052

174

503111plusmn093

299

63184plusmn10

1319

63080plusmn046

151

83125plusmn080

256

3120plusmn088

283

253249plusmn059

183

63160plusmn067

212

83127plusmn068

219

63179plusmn065

204

3177plusmn080

251

103238plusmn091

281

63269plusmn119

363

63197plusmn076

239

53235plusmn095

294

3237plusmn097

299

53314

mdash1

mdashmdash

03198plusmn005

015

33256plusmn005

015

3227plusmn058

181

05

3217

mdash1

mdashmdash

03211plusmn076

236

33214plusmn076

236

3213plusmn062

193

250

3071plusmn

049

161

830

70plusmn039

128

830

61plusmn

033

108

830

67plusmn041

132

3067plusmn040

129

100

3230plusmn074

228

83203plusmn090

281

83219plusmn040

124

83217plusmn068

211

3217plusmn069

214

5034

02plusmn093

273

63324plusmn048

144

83316plusmn093

287

83347plusmn079

235

3342plusmn085

255

2534

63plusmn10

2295

83378plusmn075

223

634

24plusmn088

258

634

21plusmn

089

259

3425plusmn093

272

103595plusmn11

2312

53526plusmn047

134

534

68plusmn00

63

180

63530plusmn074

209

3526plusmn090

256

53384plusmn062

184

3mdash

mdash0

mdashmdash

034

83plusmn060

171

3384plusmn062

184

QuantiTectR

T+qP

CRQuantitect

250

3137plusmn069

220

83145plusmn052

165

83122plusmn046

147

83135plusmn056

177

3135plusmn055

175

100

3246plusmn080

246

73272plusmn092

281

73390plusmn090

265

73303plusmn087

264

3303plusmn10

5318

503359plusmn060

179

43434plusmn232

675

53366plusmn079

235

63386plusmn12

4363

3387plusmn14

0413

253298plusmn074

224

43358plusmn118

351

53412plusmn207

608

43356plusmn13

3394

3356plusmn13

8410

103354plusmn018

054

33386plusmn061

180

23473plusmn062

179

23404plusmn047

138

3397plusmn065

192

5mdash

mdash0

3552

mdash1

mdashmdash

03552

mdash3552

mdash50

034

09plusmn036

135

836

37plusmn118

326

63297plusmn038

116

834

30plusmn15

419

234

30plusmn15

4448

250

3642plusmn046

643

83366plusmn080

239

83361plusmn

083

246

834

56plusmn19

7376

3456plusmn19

7569

100

3439plusmn234

257

834

60plusmn098

284

834

50plusmn091

265

834

50plusmn089

269

3450plusmn089

258

5036

28plusmn088

475

63507plusmn091

259

734

92plusmn10

4297

73538plusmn13

2344

3538plusmn13

2374

253597plusmn17

2230

536

28plusmn088

242

436

44plusmn078

214

536

23plusmn079

229

3623plusmn079

217

10mdash

mdash0

mdashmdash

03765plusmn079

210

23765plusmn079

210

3765plusmn079

210


(a)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CV ()lowastlowast

AverageC

tplusmnSD

CtCV

()lowastlowast

Maxim

aRT

+qP

CRMaxim

a

100

3183plusmn084

263

3138plusmn022

070

3009plusmn041

137

311plusmn049

157

311plusmn092

295

503335plusmn10

9326

83342plusmn088

264

53112plusmn064

205

83263plusmn087

265

3251plusmn

140

432

253367plusmn088

261

53277plusmn112

340

63247plusmn072

221

73297plusmn090

274

3290plusmn099

302

103716plusmn464

1249

33400plusmn14

1416

43435plusmn13

3388

53517plusmn246

684

3494plusmn263

754

53544plusmn024

068

23865

mdash1

3458plusmn12

4357

33622plusmn074

212

3555plusmn17

6496

25

mdashmdash

03314

mdash1

3347plusmn17

6526

23330plusmn17

6526

3336plusmn12

6377

250

2928plusmn058

199

829

05plusmn038

130

829

04plusmn039

135

829

12plusmn045

155

2912plusmn045

156

100

3053plusmn056

185

830

17plusmn048

159

830

31plusmn

043

143

830

34plusmn050

162

3034plusmn050

164

503154plusmn12

5397

83188plusmn13

7429

83172plusmn056

177

83171plusmn

108

334

3171plusmn

108

340

253300plusmn115

348

63238plusmn079

243

73238plusmn082

254

73256plusmn092

282

3256plusmn092

281

103308plusmn064

193

33302plusmn13

8417

43340plusmn14

6438

43317plusmn115

349

3317plusmn11

5347

503303plusmn015

045

23355

mdash1

3299

mdash1

3315plusmn028

045

3315plusmn028

085

qScriptR

T+qP

CRPerfe

cta

250

2704plusmn024

088

82675plusmn026

098

82677plusmn024

090

82685plusmn025

092

2685plusmn027

101

100

3054plusmn054

176

82984plusmn054

182

83046plusmn10

7350

83028plusmn072

236

3028plusmn079

261

503203plusmn290

905

73072plusmn089

292

83078plusmn082

267

83118plusmn15

4488

3114plusmn17

7568

253250plusmn098

302

73241plusmn

133

410

73363plusmn216

642

53285plusmn14

9452

3276plusmn14

9456

103222plusmn081

252

53287plusmn046

139

33270plusmn056

171

23260plusmn061

187

3251plusmn

069

211

53278plusmn015

045

2mdash

mdash0

3356plusmn15

5461

23318plusmn085

253

3318plusmn10

0303

250

2947plusmn022

074

829

49plusmn054

183

729

82plusmn038

129

829

60plusmn041

129

2960plusmn041

139

100

3099plusmn10

9352

83114plusmn12

6404

83102plusmn052

169

83105plusmn096

308

3105plusmn096

311

503187plusmn060

188

83137plusmn057

180

83313plusmn11

2337

83212plusmn10

8235

3212plusmn10

8335

253335plusmn299

896

73249plusmn068

211

83271plusmn

087

265

83283plusmn17

2457

3283plusmn17

2524

103300plusmn049

147

53345plusmn018

053

23298plusmn074

225

43307plusmn054

142

3307plusmn054

165

53304plusmn10

1306

23303plusmn074

223

23359

mdash1

3314plusmn067

265

3314plusmn067

203


(a)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CV ()lowastlowast

AverageC

tplusmnSD

CtCV

()lowastlowast

SSViloRT

+qP

CREx

press

100

2768plusmn059

213

82782plusmn055

197

82759plusmn029

106

82770plusmn048

172

2770plusmn048

174

502942plusmn062

211

82927plusmn054

185

82921plusmn

060

206

82930plusmn059

201

2930plusmn057

195

253038plusmn067

221

63022plusmn12

4411

82960plusmn048

162

83007plusmn080

264

3004plusmn090

300

103136plusmn049

158

73130plusmn071

227

73192plusmn078

245

63153plusmn066

210

3151plusmn

069

219

53149plusmn053

168

43163plusmn098

308

43164

mdash1

3159plusmn075

238

3157plusmn068

216

05

mdashmdash

0mdash

mdash0

3159

mdash1

3159

mdash3159

mdash100

3178plusmn053

167

830

13plusmn032

107

830

75plusmn065

211

830

89plusmn085

162

3089plusmn085

276

503296plusmn042

129

83134plusmn050

161

83149plusmn097

307

83193plusmn099

199

3193plusmn099

309

253372plusmn10

5310

73215plusmn098

306

83209plusmn022

069

63266plusmn113

228

3266plusmn11

3346

1034

44plusmn060

174

73361plusmn

046

135

53330plusmn043

129

63383plusmn071

146

3383plusmn071

209

53521plusmn

053

151

73264plusmn036

111

53299plusmn006

017

23397plusmn13

5093

3397plusmn13

5398

25

3956plusmn18

6471

334

35

13355plusmn057

169

336

24plusmn332

320

3624plusmn332

916


varia

bilitya

ndcoeffi

ciento

fvariatio

nswerec

alculated


CV=sta



100intra-assayC

V=mean

oftheC

Vso

fallruns

lowastAv

erageC


deviation

lowastlowastCV

()=

standard



Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)

(b)1stepmetho

ds

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CVlowastlowast

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Quantitect1step

502976plusmn064

216

82894plusmn055

189

82927plusmn023

079

82932plusmn047

161

2932plusmn059

106

253090plusmn088

286

82984plusmn10

9365

83098plusmn076

245

83057plusmn091

299

3057plusmn10

3202

103172plusmn10

4326

73205plusmn10

6332

73230plusmn061

188

83203plusmn090

282

3204plusmn090

336

53334plusmn12

0360

63238plusmn074

228

73285plusmn084

255

73286plusmn092

281

3283plusmn096

281

25

3251plusmn

060

185

43390plusmn033

096

43445plusmn15

2442

53362plusmn082

241

3368plusmn12

7377

13338plusmn096

288

43203plusmn026

080

33292plusmn047

143

53277plusmn056

170

3285plusmn079

242

5030

92plusmn047

152

83123plusmn12

9040

83111plusmn13

2041

83108plusmn13

8043

3108plusmn043

138

253177plusmn073

229

83192plusmn14

9048

83191plusmn

093

030

83187plusmn15

7050

3187plusmn051

161

103260plusmn047

144

83281plusmn

061

187

53249plusmn065

199

83263plusmn058

177

3263plusmn057

175

53369plusmn085

253

73382plusmn291

098

53328plusmn250

083

63360plusmn265

089

3357plusmn086

257

25

3436plusmn073

214

53365plusmn18

8063

23327plusmn18

8062

33376plusmn19

6066

3380plusmn080

236

134

55plusmn17

7513

334

97plusmn117

041

53392plusmn341

116

134

48plusmn324

111

3445plusmn13

7397


(b)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CVlowastlowast

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

RotorG

ene1

step

1000

2640plusmn045

169

82374plusmn040

167

82701plusmn

118

438

82572plusmn16

2258

2572plusmn16

2632

500

2749plusmn046

166

82429plusmn034

139

82742plusmn10

4378

82640plusmn16

6228

2640plusmn16

6628

250

2873plusmn076

265

82490plusmn049

196

82927plusmn060

204

72756plusmn209

222

2756plusmn209

758

100

3070plusmn021

067

32550

mdash1

3119plusmn066

211

23000plusmn224

139

3000plusmn224

746

503031

mdash1

mdashmdash

0mdash

mdash0

3031

mdash3031

mdash25

3112plusmn050

161

2mdash

mdash0

mdashmdash

03212plusmn050

161

3212plusmn050

161

5028

87plusmn099

341

829

25plusmn074

252

82797plusmn065

233

828

70plusmn079

276

2870plusmn094

329

2529

49plusmn10

1344

830

36plusmn070

229

828

57plusmn039

136

829

47plusmn070

236

2947plusmn10

3350

1030

29plusmn12

0398

830

61plusmn

144

470

828

76plusmn040

139

829

89plusmn10

1336

2989plusmn13

4450

530

82plusmn10

6343

63147plusmn13

0412

629

90plusmn040

135

830

73plusmn092

297

3065plusmn11

2366

25

3087plusmn114

368

63169plusmn057

181

529

10plusmn037

126

530

55plusmn069

225

3057plusmn13

1429

13122

mdash1

3153

mdash1

3201plusmn

044

137

53159plusmn044

137

3183plusmn048

151

Verso1step

500

2650plusmn018

068

82656plusmn014

053

82679plusmn023

085

82661plusmn

018

069

2661plusmn

022

082

250

2743plusmn018

065

82796plusmn027

098

82789plusmn036

127

82776plusmn027

097

2776plusmn036

130

100

2894plusmn050

172

82942plusmn053

180

82991plusmn

118

396

82943plusmn074

249

2943plusmn087

295

503049plusmn044

143

73138plusmn086

274

83129plusmn15

2487

83105plusmn094

301

3108plusmn10

9350

253282plusmn114

346

73290plusmn052

157

53250plusmn092

282

73274plusmn086

262

3272plusmn090

274

103342plusmn097

291

33522plusmn10

5299

23442

mdash1

3435plusmn10

1295

3419plusmn118

344

250

2939plusmn048

164

829

40plusmn023

079

829

10plusmn053

183

829

20plusmn042

142

2930plusmn044

150

100

3055plusmn025

080

830

32plusmn038

125

830

02plusmn024

082

630

29plusmn029

096

3032plusmn036

119

503116plusmn050

159

83110plusmn052

168

830

87plusmn042

135

73104plusmn048

154

3105plusmn048

153

253171plusmn

032

102

73161plusmn

042

134

83135plusmn058

184

83156plusmn044

140

3155plusmn046

147

103186plusmn054

169

73176plusmn029

091

73165plusmn064

201

43176plusmn049

154

3177plusmn046

145

53309plusmn098

297

53313

13343plusmn074

223

43322plusmn086

260

3326plusmn080

239


(b)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CVlowastlowast

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

qScript1

step

1000

2324plusmn012

053

82297plusmn024

103

82321plusmn

009

037

82314plusmn015

064

2314plusmn020

086

750

2364plusmn021

088

82329plusmn008

035

82352plusmn028

121

82348plusmn019

081

2348plusmn025

106

500

2446plusmn032

132

82406plusmn024

098

82407plusmn029

122

82419plusmn028

117

2419plusmn033

137

250

2538plusmn027

107

82493plusmn026

105

82514plusmn034

137

82515plusmn029

116

2515plusmn034

135

100

2626plusmn031

116

82579plusmn024

093

72595plusmn047

180

52600plusmn034

130

2602plusmn038

146

50mdash

mdash0

mdashmdash

02645plusmn018

070

22645plusmn018

070

2645plusmn018

070

2500

2813plusmn083

294

82778plusmn045

160

826

32plusmn030

113

82741plusmn

052

189

2741plusmn

097

353

1000

2878plusmn009

033

829

40plusmn15

8539

824

89plusmn030

122

82769plusmn066

231

2769plusmn223

805

750

2908plusmn024

082

828

92plusmn025

085

828

75plusmn011

038

828

92plusmn020

068

2892plusmn024

084

500

3069plusmn048

157

729

76plusmn055

186

82763plusmn056

201

529

36plusmn053

181

2966plusmn12

6424

250

3122plusmn023

073

730

05plusmn037

124

8mdash

mdash0

3064plusmn030

099

3060plusmn067

220

100

mdashmdash

030

00

mdash1

mdashmdash

030

00

mdash30

00

mdash

SSIIIP

latinium

1step

1000

2479plusmn024

097

82381plusmn

021

089

82446plusmn015

060

82435plusmn020

082

2435plusmn046

188

750

2518plusmn024

095

82430plusmn012

051

82497plusmn010

042

82482plusmn016

063

2482plusmn041

167

500

2666plusmn082

307

82536plusmn023

092

82619plusmn024

092

82607plusmn043

164

2607plusmn073

282

250

2866plusmn086

301

82838plusmn19

1672

82897plusmn059

205

82867plusmn112

393

2867plusmn12

2427

100

2917plusmn053

180

82778plusmn049

175

82816plusmn053

189

72837plusmn052

182

2838plusmn078

276

503095plusmn043

139

22911plusmn067

231

42993plusmn111

370

33000plusmn074

247

2979plusmn10

3347

250

2627plusmn026

099

826

70plusmn027

102

826

67plusmn042

156

826

55plusmn037

119

2655plusmn037

139

100

2722plusmn068

248

828

02plusmn039

138

82774plusmn034

121

82766plusmn058

169

2766plusmn058

209

502756plusmn053

194

828

26plusmn041

147

828

33plusmn040

141

828

05plusmn056

161

2805plusmn056

200

252787plusmn069

247

829

34plusmn055

189

828

58plusmn041

145

828

59plusmn082

194

2859plusmn082

285

1028

75plusmn078

270

629

44plusmn055

186

529

08plusmn033

114

729

07plusmn060

190

2907plusmn060

207

528

82plusmn015

053

428

71plusmn

021

072

328

88plusmn032

112

428

81plusmn

023

079

2881plusmn

023

079

Theintra-a

ndinterassay

varia

bilityandcoeffi

ciento

fvariatio

nswerec

alculated


=standard

deviationof



100intra-assayCV

=mean

oftheC

Vso

fallruns

lowastAv

erageC


deviation

lowastlowastCV

()=

standard



Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)






RotorGene QMDx

Mx3005P






4 Discussion












5 Conclusion



Abbreviations




Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




















Table3Com

paris

onof

qRT-PC

RSY

BRGreen

(one-step)

kits

Results

offived

ifferentq

RT-PCR

kitson

arange

ofseventriplicated

ilutio

nsof

EBLV

-1in

vitro

RNAtranscrip

tsTestingof

different

qRT-PC

Rkits(one-step)

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Quantitect-1ste

pRo

torG

ene-1step

Verso-1step

SSIIIP

latinium-1ste

pqScript-1

step

AverageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowastAv

erageC

tlowastlowastlowast

CV(

)lowast

106

2010

61666plusmn008

045

1715plusmn005

029

1699plusmn006

038

1452plusmn019

032

1347plusmn003

019

1779plusmn024

132

1588plusmn022

136

1834plusmn032

172

1622plusmn004

022

1848plusmn004

020

105

2010

52006plusmn010

052

2062plusmn004

017

2040plusmn011

052

1790plusmn021

118

1655plusmn003

019

2113plusmn045

212

1942plusmn020

104

2160plusmn015

071

1964plusmn041

211

2238plusmn015

068

104

2010

42358plusmn014

058

2391plusmn

008

033

2382plusmn020

083

2143plusmn012

057

1986plusmn004

018

2460plusmn020

083

2287plusmn016

069

2517plusmn017

069

2308plusmn027

116

2611plusmn010

038

103

2010

32670plusmn024

091

2745plusmn028

101

2693plusmn003

011

2490plusmn023

092

2313plusmn016

067

2795plusmn026

093

2634plusmn033

124

2863plusmn030

106

2614plusmn010

038

2954plusmn025

086

102

2010

23042plusmn008

026

23lowastlowast

3019plusmn060

198

03lowastlowast

03lowastlowast

3198plusmn017

053

2977plusmn039

132

3174plusmn079

250

2893plusmn008

028

3107plusmn037

118

101

2010

13324plusmn029

087

13lowastlowast

3327plusmn15

3460

03lowastlowast

03lowastlowast

3434plusmn10

3299

3284plusmn044

133

23lowastlowast

2991plusmn

010

033

03lowastlowast

12

03lowastlowast

13lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

03lowastlowast

23lowastlowast

03lowastlowast

03lowastlowast

Efficiency

98

96

100

94

104

97

94

95

100

87

1198772

0998

0999

0999

0998

0999

099

6099

8099

7099

6099

8

119910-in

tercept

3689

3767

3699

3529

3278

3815

3680

3893

3893

4074

lowastCV

()=

standard


CVwas

notcalculated

whensomeo

fthe

parallelsfailedto

amplify


bero

fpositivesnu

mbero

fexp

erim

ents


erageC


deviation


Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)


Table4Detectio

nlim

itof

PCR(LOD95)for

two-ste

pandon

e-ste

pSY

BRGreen

RT-PCR

Two-ste

pRT

-qPC

RIScriptR

T+qP

CRSsoF

ast

BioR

adQuantiTectR

T+qP

CRQuantitect

QIAGEN

Maxim

aRT+qP

CRMaxim

aFE

RMEN

TAS

SSViloRT

+qP

CREx

press

INVITRO

GEN

qScriptR

T+qP

CRPerfe

cta

QUA

NTA

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

11055

171

8678

3941

2188

4488

44160

8076

3845

2267

34One-stepqR

T-PC

RQuantitect-1ste

pQIAGEN

RotorG

ene-1step

QIAGEN

Verso-1step

FERM

ENTA

SSSIIIP

latinium-1ste

pIN

VITRO

GEN

qScript-1

step

QUA

NTA

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

2040

497

994

127

254

321

641

406

812

2244

1530

101

202

50100

981

1962

lowastAllLO

D95results

werec

arrie

dou

tusin

gProb

itmod

ellowastAllPC

Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)


Table5Analysis

ofreprod

ucibilityof

one-ste

pandtwo-ste

pRT

-PCR

(a)

show

sfor

each

two-ste

pkittheintra-a

ndinterassay

reprod


nsof

EBLV

-1teste

deighttim

esin

each

ofthreeind

ependent

runs


nd(b)sho

wsthe


ucibilityfora

llteste

don

e-ste

pkits

(a)2-ste

pmetho

ds

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CV ()lowastlowast

AverageC

tplusmnSD

CtCV

()lowastlowast

IScriptR

T+qP

CRSsoF

ast

100

3046plusmn041

135

83008plusmn057

190

82982plusmn041

138

83012plusmn046

154

3012plusmn052

174

503111plusmn093

299

63184plusmn10

1319

63080plusmn046

151

83125plusmn080

256

3120plusmn088

283

253249plusmn059

183

63160plusmn067

212

83127plusmn068

219

63179plusmn065

204

3177plusmn080

251

103238plusmn091

281

63269plusmn119

363

63197plusmn076

239

53235plusmn095

294

3237plusmn097

299

53314

mdash1

mdashmdash

03198plusmn005

015

33256plusmn005

015

3227plusmn058

181

05

3217

mdash1

mdashmdash

03211plusmn076

236

33214plusmn076

236

3213plusmn062

193

250

3071plusmn

049

161

830

70plusmn039

128

830

61plusmn

033

108

830

67plusmn041

132

3067plusmn040

129

100

3230plusmn074

228

83203plusmn090

281

83219plusmn040

124

83217plusmn068

211

3217plusmn069

214

5034

02plusmn093

273

63324plusmn048

144

83316plusmn093

287

83347plusmn079

235

3342plusmn085

255

2534

63plusmn10

2295

83378plusmn075

223

634

24plusmn088

258

634

21plusmn

089

259

3425plusmn093

272

103595plusmn11

2312

53526plusmn047

134

534

68plusmn00

63

180

63530plusmn074

209

3526plusmn090

256

53384plusmn062

184

3mdash

mdash0

mdashmdash

034

83plusmn060

171

3384plusmn062

184

QuantiTectR

T+qP

CRQuantitect

250

3137plusmn069

220

83145plusmn052

165

83122plusmn046

147

83135plusmn056

177

3135plusmn055

175

100

3246plusmn080

246

73272plusmn092

281

73390plusmn090

265

73303plusmn087

264

3303plusmn10

5318

503359plusmn060

179

43434plusmn232

675

53366plusmn079

235

63386plusmn12

4363

3387plusmn14

0413

253298plusmn074

224

43358plusmn118

351

53412plusmn207

608

43356plusmn13

3394

3356plusmn13

8410

103354plusmn018

054

33386plusmn061

180

23473plusmn062

179

23404plusmn047

138

3397plusmn065

192

5mdash

mdash0

3552

mdash1

mdashmdash

03552

mdash3552

mdash50

034

09plusmn036

135

836

37plusmn118

326

63297plusmn038

116

834

30plusmn15

419

234

30plusmn15

4448

250

3642plusmn046

643

83366plusmn080

239

83361plusmn

083

246

834

56plusmn19

7376

3456plusmn19

7569

100

3439plusmn234

257

834

60plusmn098

284

834

50plusmn091

265

834

50plusmn089

269

3450plusmn089

258

5036

28plusmn088

475

63507plusmn091

259

734

92plusmn10

4297

73538plusmn13

2344

3538plusmn13

2374

253597plusmn17

2230

536

28plusmn088

242

436

44plusmn078

214

536

23plusmn079

229

3623plusmn079

217

10mdash

mdash0

mdashmdash

03765plusmn079

210

23765plusmn079

210

3765plusmn079

210


(a)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CV ()lowastlowast

AverageC

tplusmnSD

CtCV

()lowastlowast

Maxim

aRT

+qP

CRMaxim

a

100

3183plusmn084

263

3138plusmn022

070

3009plusmn041

137

311plusmn049

157

311plusmn092

295

503335plusmn10

9326

83342plusmn088

264

53112plusmn064

205

83263plusmn087

265

3251plusmn

140

432

253367plusmn088

261

53277plusmn112

340

63247plusmn072

221

73297plusmn090

274

3290plusmn099

302

103716plusmn464

1249

33400plusmn14

1416

43435plusmn13

3388

53517plusmn246

684

3494plusmn263

754

53544plusmn024

068

23865

mdash1

3458plusmn12

4357

33622plusmn074

212

3555plusmn17

6496

25

mdashmdash

03314

mdash1

3347plusmn17

6526

23330plusmn17

6526

3336plusmn12

6377

250

2928plusmn058

199

829

05plusmn038

130

829

04plusmn039

135

829

12plusmn045

155

2912plusmn045

156

100

3053plusmn056

185

830

17plusmn048

159

830

31plusmn

043

143

830

34plusmn050

162

3034plusmn050

164

503154plusmn12

5397

83188plusmn13

7429

83172plusmn056

177

83171plusmn

108

334

3171plusmn

108

340

253300plusmn115

348

63238plusmn079

243

73238plusmn082

254

73256plusmn092

282

3256plusmn092

281

103308plusmn064

193

33302plusmn13

8417

43340plusmn14

6438

43317plusmn115

349

3317plusmn11

5347

503303plusmn015

045

23355

mdash1

3299

mdash1

3315plusmn028

045

3315plusmn028

085

qScriptR

T+qP

CRPerfe

cta

250

2704plusmn024

088

82675plusmn026

098

82677plusmn024

090

82685plusmn025

092

2685plusmn027

101

100

3054plusmn054

176

82984plusmn054

182

83046plusmn10

7350

83028plusmn072

236

3028plusmn079

261

503203plusmn290

905

73072plusmn089

292

83078plusmn082

267

83118plusmn15

4488

3114plusmn17

7568

253250plusmn098

302

73241plusmn

133

410

73363plusmn216

642

53285plusmn14

9452

3276plusmn14

9456

103222plusmn081

252

53287plusmn046

139

33270plusmn056

171

23260plusmn061

187

3251plusmn

069

211

53278plusmn015

045

2mdash

mdash0

3356plusmn15

5461

23318plusmn085

253

3318plusmn10

0303

250

2947plusmn022

074

829

49plusmn054

183

729

82plusmn038

129

829

60plusmn041

129

2960plusmn041

139

100

3099plusmn10

9352

83114plusmn12

6404

83102plusmn052

169

83105plusmn096

308

3105plusmn096

311

503187plusmn060

188

83137plusmn057

180

83313plusmn11

2337

83212plusmn10

8235

3212plusmn10

8335

253335plusmn299

896

73249plusmn068

211

83271plusmn

087

265

83283plusmn17

2457

3283plusmn17

2524

103300plusmn049

147

53345plusmn018

053

23298plusmn074

225

43307plusmn054

142

3307plusmn054

165

53304plusmn10

1306

23303plusmn074

223

23359

mdash1

3314plusmn067

265

3314plusmn067

203


(a)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CV ()lowastlowast

AverageC

tplusmnSD

CtCV

()lowastlowast

SSViloRT

+qP

CREx

press

100

2768plusmn059

213

82782plusmn055

197

82759plusmn029

106

82770plusmn048

172

2770plusmn048

174

502942plusmn062

211

82927plusmn054

185

82921plusmn

060

206

82930plusmn059

201

2930plusmn057

195

253038plusmn067

221

63022plusmn12

4411

82960plusmn048

162

83007plusmn080

264

3004plusmn090

300

103136plusmn049

158

73130plusmn071

227

73192plusmn078

245

63153plusmn066

210

3151plusmn

069

219

53149plusmn053

168

43163plusmn098

308

43164

mdash1

3159plusmn075

238

3157plusmn068

216

05

mdashmdash

0mdash

mdash0

3159

mdash1

3159

mdash3159

mdash100

3178plusmn053

167

830

13plusmn032

107

830

75plusmn065

211

830

89plusmn085

162

3089plusmn085

276

503296plusmn042

129

83134plusmn050

161

83149plusmn097

307

83193plusmn099

199

3193plusmn099

309

253372plusmn10

5310

73215plusmn098

306

83209plusmn022

069

63266plusmn113

228

3266plusmn11

3346

1034

44plusmn060

174

73361plusmn

046

135

53330plusmn043

129

63383plusmn071

146

3383plusmn071

209

53521plusmn

053

151

73264plusmn036

111

53299plusmn006

017

23397plusmn13

5093

3397plusmn13

5398

25

3956plusmn18

6471

334

35

13355plusmn057

169

336

24plusmn332

320

3624plusmn332

916


varia

bilitya

ndcoeffi

ciento

fvariatio

nswerec

alculated


CV=sta



100intra-assayC

V=mean

oftheC

Vso

fallruns

lowastAv

erageC


deviation

lowastlowastCV

()=

standard



Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)

(b)1stepmetho

ds

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CVlowastlowast

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Quantitect1step

502976plusmn064

216

82894plusmn055

189

82927plusmn023

079

82932plusmn047

161

2932plusmn059

106

253090plusmn088

286

82984plusmn10

9365

83098plusmn076

245

83057plusmn091

299

3057plusmn10

3202

103172plusmn10

4326

73205plusmn10

6332

73230plusmn061

188

83203plusmn090

282

3204plusmn090

336

53334plusmn12

0360

63238plusmn074

228

73285plusmn084

255

73286plusmn092

281

3283plusmn096

281

25

3251plusmn

060

185

43390plusmn033

096

43445plusmn15

2442

53362plusmn082

241

3368plusmn12

7377

13338plusmn096

288

43203plusmn026

080

33292plusmn047

143

53277plusmn056

170

3285plusmn079

242

5030

92plusmn047

152

83123plusmn12

9040

83111plusmn13

2041

83108plusmn13

8043

3108plusmn043

138

253177plusmn073

229

83192plusmn14

9048

83191plusmn

093

030

83187plusmn15

7050

3187plusmn051

161

103260plusmn047

144

83281plusmn

061

187

53249plusmn065

199

83263plusmn058

177

3263plusmn057

175

53369plusmn085

253

73382plusmn291

098

53328plusmn250

083

63360plusmn265

089

3357plusmn086

257

25

3436plusmn073

214

53365plusmn18

8063

23327plusmn18

8062

33376plusmn19

6066

3380plusmn080

236

134

55plusmn17

7513

334

97plusmn117

041

53392plusmn341

116

134

48plusmn324

111

3445plusmn13

7397


(b)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CVlowastlowast

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

RotorG

ene1

step

1000

2640plusmn045

169

82374plusmn040

167

82701plusmn

118

438

82572plusmn16

2258

2572plusmn16

2632

500

2749plusmn046

166

82429plusmn034

139

82742plusmn10

4378

82640plusmn16

6228

2640plusmn16

6628

250

2873plusmn076

265

82490plusmn049

196

82927plusmn060

204

72756plusmn209

222

2756plusmn209

758

100

3070plusmn021

067

32550

mdash1

3119plusmn066

211

23000plusmn224

139

3000plusmn224

746

503031

mdash1

mdashmdash

0mdash

mdash0

3031

mdash3031

mdash25

3112plusmn050

161

2mdash

mdash0

mdashmdash

03212plusmn050

161

3212plusmn050

161

5028

87plusmn099

341

829

25plusmn074

252

82797plusmn065

233

828

70plusmn079

276

2870plusmn094

329

2529

49plusmn10

1344

830

36plusmn070

229

828

57plusmn039

136

829

47plusmn070

236

2947plusmn10

3350

1030

29plusmn12

0398

830

61plusmn

144

470

828

76plusmn040

139

829

89plusmn10

1336

2989plusmn13

4450

530

82plusmn10

6343

63147plusmn13

0412

629

90plusmn040

135

830

73plusmn092

297

3065plusmn11

2366

25

3087plusmn114

368

63169plusmn057

181

529

10plusmn037

126

530

55plusmn069

225

3057plusmn13

1429

13122

mdash1

3153

mdash1

3201plusmn

044

137

53159plusmn044

137

3183plusmn048

151

Verso1step

500

2650plusmn018

068

82656plusmn014

053

82679plusmn023

085

82661plusmn

018

069

2661plusmn

022

082

250

2743plusmn018

065

82796plusmn027

098

82789plusmn036

127

82776plusmn027

097

2776plusmn036

130

100

2894plusmn050

172

82942plusmn053

180

82991plusmn

118

396

82943plusmn074

249

2943plusmn087

295

503049plusmn044

143

73138plusmn086

274

83129plusmn15

2487

83105plusmn094

301

3108plusmn10

9350

253282plusmn114

346

73290plusmn052

157

53250plusmn092

282

73274plusmn086

262

3272plusmn090

274

103342plusmn097

291

33522plusmn10

5299

23442

mdash1

3435plusmn10

1295

3419plusmn118

344

250

2939plusmn048

164

829

40plusmn023

079

829

10plusmn053

183

829

20plusmn042

142

2930plusmn044

150

100

3055plusmn025

080

830

32plusmn038

125

830

02plusmn024

082

630

29plusmn029

096

3032plusmn036

119

503116plusmn050

159

83110plusmn052

168

830

87plusmn042

135

73104plusmn048

154

3105plusmn048

153

253171plusmn

032

102

73161plusmn

042

134

83135plusmn058

184

83156plusmn044

140

3155plusmn046

147

103186plusmn054

169

73176plusmn029

091

73165plusmn064

201

43176plusmn049

154

3177plusmn046

145

53309plusmn098

297

53313

13343plusmn074

223

43322plusmn086

260

3326plusmn080

239


(b)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CVlowastlowast

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

qScript1

step

1000

2324plusmn012

053

82297plusmn024

103

82321plusmn

009

037

82314plusmn015

064

2314plusmn020

086

750

2364plusmn021

088

82329plusmn008

035

82352plusmn028

121

82348plusmn019

081

2348plusmn025

106

500

2446plusmn032

132

82406plusmn024

098

82407plusmn029

122

82419plusmn028

117

2419plusmn033

137

250

2538plusmn027

107

82493plusmn026

105

82514plusmn034

137

82515plusmn029

116

2515plusmn034

135

100

2626plusmn031

116

82579plusmn024

093

72595plusmn047

180

52600plusmn034

130

2602plusmn038

146

50mdash

mdash0

mdashmdash

02645plusmn018

070

22645plusmn018

070

2645plusmn018

070

2500

2813plusmn083

294

82778plusmn045

160

826

32plusmn030

113

82741plusmn

052

189

2741plusmn

097

353

1000

2878plusmn009

033

829

40plusmn15

8539

824

89plusmn030

122

82769plusmn066

231

2769plusmn223

805

750

2908plusmn024

082

828

92plusmn025

085

828

75plusmn011

038

828

92plusmn020

068

2892plusmn024

084

500

3069plusmn048

157

729

76plusmn055

186

82763plusmn056

201

529

36plusmn053

181

2966plusmn12

6424

250

3122plusmn023

073

730

05plusmn037

124

8mdash

mdash0

3064plusmn030

099

3060plusmn067

220

100

mdashmdash

030

00

mdash1

mdashmdash

030

00

mdash30

00

mdash

SSIIIP

latinium

1step

1000

2479plusmn024

097

82381plusmn

021

089

82446plusmn015

060

82435plusmn020

082

2435plusmn046

188

750

2518plusmn024

095

82430plusmn012

051

82497plusmn010

042

82482plusmn016

063

2482plusmn041

167

500

2666plusmn082

307

82536plusmn023

092

82619plusmn024

092

82607plusmn043

164

2607plusmn073

282

250

2866plusmn086

301

82838plusmn19

1672

82897plusmn059

205

82867plusmn112

393

2867plusmn12

2427

100

2917plusmn053

180

82778plusmn049

175

82816plusmn053

189

72837plusmn052

182

2838plusmn078

276

503095plusmn043

139

22911plusmn067

231

42993plusmn111

370

33000plusmn074

247

2979plusmn10

3347

250

2627plusmn026

099

826

70plusmn027

102

826

67plusmn042

156

826

55plusmn037

119

2655plusmn037

139

100

2722plusmn068

248

828

02plusmn039

138

82774plusmn034

121

82766plusmn058

169

2766plusmn058

209

502756plusmn053

194

828

26plusmn041

147

828

33plusmn040

141

828

05plusmn056

161

2805plusmn056

200

252787plusmn069

247

829

34plusmn055

189

828

58plusmn041

145

828

59plusmn082

194

2859plusmn082

285

1028

75plusmn078

270

629

44plusmn055

186

529

08plusmn033

114

729

07plusmn060

190

2907plusmn060

207

528

82plusmn015

053

428

71plusmn

021

072

328

88plusmn032

112

428

81plusmn

023

079

2881plusmn

023

079

Theintra-a

ndinterassay

varia

bilityandcoeffi

ciento

fvariatio

nswerec

alculated


=standard

deviationof



100intra-assayCV

=mean

oftheC

Vso

fallruns

lowastAv

erageC


deviation

lowastlowastCV

()=

standard



Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)






RotorGene QMDx

Mx3005P






4 Discussion












5 Conclusion



Abbreviations




Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table4Detectio

nlim

itof

PCR(LOD95)for

two-ste

pandon

e-ste

pSY

BRGreen

RT-PCR

Two-ste

pRT

-qPC

RIScriptR

T+qP

CRSsoF

ast

BioR

adQuantiTectR

T+qP

CRQuantitect

QIAGEN

Maxim

aRT+qP

CRMaxim

aFE

RMEN

TAS

SSViloRT

+qP

CREx

press

INVITRO

GEN

qScriptR

T+qP

CRPerfe

cta

QUA

NTA

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

11055

171

8678

3941

2188

4488

44160

8076

3845

2267

34One-stepqR

T-PC

RQuantitect-1ste

pQIAGEN

RotorG

ene-1step

QIAGEN

Verso-1step

FERM

ENTA

SSSIIIP

latinium-1ste

pIN

VITRO

GEN

qScript-1

step

QUA

NTA

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

Num

bero

fcopiessam

ple

Num

bero

fcopiesrxn

2040

497

994

127

254

321

641

406

812

2244

1530

101

202

50100

981

1962

lowastAllLO

D95results

werec

arrie

dou

tusin

gProb

itmod

ellowastAllPC

Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)


Table5Analysis

ofreprod

ucibilityof

one-ste

pandtwo-ste

pRT

-PCR

(a)

show

sfor

each

two-ste

pkittheintra-a

ndinterassay

reprod


nsof

EBLV

-1teste

deighttim

esin

each

ofthreeind

ependent

runs


nd(b)sho

wsthe


ucibilityfora

llteste

don

e-ste

pkits

(a)2-ste

pmetho

ds

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CV ()lowastlowast

AverageC

tplusmnSD

CtCV

()lowastlowast

IScriptR

T+qP

CRSsoF

ast

100

3046plusmn041

135

83008plusmn057

190

82982plusmn041

138

83012plusmn046

154

3012plusmn052

174

503111plusmn093

299

63184plusmn10

1319

63080plusmn046

151

83125plusmn080

256

3120plusmn088

283

253249plusmn059

183

63160plusmn067

212

83127plusmn068

219

63179plusmn065

204

3177plusmn080

251

103238plusmn091

281

63269plusmn119

363

63197plusmn076

239

53235plusmn095

294

3237plusmn097

299

53314

mdash1

mdashmdash

03198plusmn005

015

33256plusmn005

015

3227plusmn058

181

05

3217

mdash1

mdashmdash

03211plusmn076

236

33214plusmn076

236

3213plusmn062

193

250

3071plusmn

049

161

830

70plusmn039

128

830

61plusmn

033

108

830

67plusmn041

132

3067plusmn040

129

100

3230plusmn074

228

83203plusmn090

281

83219plusmn040

124

83217plusmn068

211

3217plusmn069

214

5034

02plusmn093

273

63324plusmn048

144

83316plusmn093

287

83347plusmn079

235

3342plusmn085

255

2534

63plusmn10

2295

83378plusmn075

223

634

24plusmn088

258

634

21plusmn

089

259

3425plusmn093

272

103595plusmn11

2312

53526plusmn047

134

534

68plusmn00

63

180

63530plusmn074

209

3526plusmn090

256

53384plusmn062

184

3mdash

mdash0

mdashmdash

034

83plusmn060

171

3384plusmn062

184

QuantiTectR

T+qP

CRQuantitect

250

3137plusmn069

220

83145plusmn052

165

83122plusmn046

147

83135plusmn056

177

3135plusmn055

175

100

3246plusmn080

246

73272plusmn092

281

73390plusmn090

265

73303plusmn087

264

3303plusmn10

5318

503359plusmn060

179

43434plusmn232

675

53366plusmn079

235

63386plusmn12

4363

3387plusmn14

0413

253298plusmn074

224

43358plusmn118

351

53412plusmn207

608

43356plusmn13

3394

3356plusmn13

8410

103354plusmn018

054

33386plusmn061

180

23473plusmn062

179

23404plusmn047

138

3397plusmn065

192

5mdash

mdash0

3552

mdash1

mdashmdash

03552

mdash3552

mdash50

034

09plusmn036

135

836

37plusmn118

326

63297plusmn038

116

834

30plusmn15

419

234

30plusmn15

4448

250

3642plusmn046

643

83366plusmn080

239

83361plusmn

083

246

834

56plusmn19

7376

3456plusmn19

7569

100

3439plusmn234

257

834

60plusmn098

284

834

50plusmn091

265

834

50plusmn089

269

3450plusmn089

258

5036

28plusmn088

475

63507plusmn091

259

734

92plusmn10

4297

73538plusmn13

2344

3538plusmn13

2374

253597plusmn17

2230

536

28plusmn088

242

436

44plusmn078

214

536

23plusmn079

229

3623plusmn079

217

10mdash

mdash0

mdashmdash

03765plusmn079

210

23765plusmn079

210

3765plusmn079

210


(a)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CV ()lowastlowast

AverageC

tplusmnSD

CtCV

()lowastlowast

Maxim

aRT

+qP

CRMaxim

a

100

3183plusmn084

263

3138plusmn022

070

3009plusmn041

137

311plusmn049

157

311plusmn092

295

503335plusmn10

9326

83342plusmn088

264

53112plusmn064

205

83263plusmn087

265

3251plusmn

140

432

253367plusmn088

261

53277plusmn112

340

63247plusmn072

221

73297plusmn090

274

3290plusmn099

302

103716plusmn464

1249

33400plusmn14

1416

43435plusmn13

3388

53517plusmn246

684

3494plusmn263

754

53544plusmn024

068

23865

mdash1

3458plusmn12

4357

33622plusmn074

212

3555plusmn17

6496

25

mdashmdash

03314

mdash1

3347plusmn17

6526

23330plusmn17

6526

3336plusmn12

6377

250

2928plusmn058

199

829

05plusmn038

130

829

04plusmn039

135

829

12plusmn045

155

2912plusmn045

156

100

3053plusmn056

185

830

17plusmn048

159

830

31plusmn

043

143

830

34plusmn050

162

3034plusmn050

164

503154plusmn12

5397

83188plusmn13

7429

83172plusmn056

177

83171plusmn

108

334

3171plusmn

108

340

253300plusmn115

348

63238plusmn079

243

73238plusmn082

254

73256plusmn092

282

3256plusmn092

281

103308plusmn064

193

33302plusmn13

8417

43340plusmn14

6438

43317plusmn115

349

3317plusmn11

5347

503303plusmn015

045

23355

mdash1

3299

mdash1

3315plusmn028

045

3315plusmn028

085

qScriptR

T+qP

CRPerfe

cta

250

2704plusmn024

088

82675plusmn026

098

82677plusmn024

090

82685plusmn025

092

2685plusmn027

101

100

3054plusmn054

176

82984plusmn054

182

83046plusmn10

7350

83028plusmn072

236

3028plusmn079

261

503203plusmn290

905

73072plusmn089

292

83078plusmn082

267

83118plusmn15

4488

3114plusmn17

7568

253250plusmn098

302

73241plusmn

133

410

73363plusmn216

642

53285plusmn14

9452

3276plusmn14

9456

103222plusmn081

252

53287plusmn046

139

33270plusmn056

171

23260plusmn061

187

3251plusmn

069

211

53278plusmn015

045

2mdash

mdash0

3356plusmn15

5461

23318plusmn085

253

3318plusmn10

0303

250

2947plusmn022

074

829

49plusmn054

183

729

82plusmn038

129

829

60plusmn041

129

2960plusmn041

139

100

3099plusmn10

9352

83114plusmn12

6404

83102plusmn052

169

83105plusmn096

308

3105plusmn096

311

503187plusmn060

188

83137plusmn057

180

83313plusmn11

2337

83212plusmn10

8235

3212plusmn10

8335

253335plusmn299

896

73249plusmn068

211

83271plusmn

087

265

83283plusmn17

2457

3283plusmn17

2524

103300plusmn049

147

53345plusmn018

053

23298plusmn074

225

43307plusmn054

142

3307plusmn054

165

53304plusmn10

1306

23303plusmn074

223

23359

mdash1

3314plusmn067

265

3314plusmn067

203


(a)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CV ()lowastlowast

AverageC

tplusmnSD

CtCV

()lowastlowast

SSViloRT

+qP

CREx

press

100

2768plusmn059

213

82782plusmn055

197

82759plusmn029

106

82770plusmn048

172

2770plusmn048

174

502942plusmn062

211

82927plusmn054

185

82921plusmn

060

206

82930plusmn059

201

2930plusmn057

195

253038plusmn067

221

63022plusmn12

4411

82960plusmn048

162

83007plusmn080

264

3004plusmn090

300

103136plusmn049

158

73130plusmn071

227

73192plusmn078

245

63153plusmn066

210

3151plusmn

069

219

53149plusmn053

168

43163plusmn098

308

43164

mdash1

3159plusmn075

238

3157plusmn068

216

05

mdashmdash

0mdash

mdash0

3159

mdash1

3159

mdash3159

mdash100

3178plusmn053

167

830

13plusmn032

107

830

75plusmn065

211

830

89plusmn085

162

3089plusmn085

276

503296plusmn042

129

83134plusmn050

161

83149plusmn097

307

83193plusmn099

199

3193plusmn099

309

253372plusmn10

5310

73215plusmn098

306

83209plusmn022

069

63266plusmn113

228

3266plusmn11

3346

1034

44plusmn060

174

73361plusmn

046

135

53330plusmn043

129

63383plusmn071

146

3383plusmn071

209

53521plusmn

053

151

73264plusmn036

111

53299plusmn006

017

23397plusmn13

5093

3397plusmn13

5398

25

3956plusmn18

6471

334

35

13355plusmn057

169

336

24plusmn332

320

3624plusmn332

916


varia

bilitya

ndcoeffi

ciento

fvariatio

nswerec

alculated


CV=sta



100intra-assayC

V=mean

oftheC

Vso

fallruns

lowastAv

erageC


deviation

lowastlowastCV

()=

standard



Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)

(b)1stepmetho

ds

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CVlowastlowast

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Quantitect1step

502976plusmn064

216

82894plusmn055

189

82927plusmn023

079

82932plusmn047

161

2932plusmn059

106

253090plusmn088

286

82984plusmn10

9365

83098plusmn076

245

83057plusmn091

299

3057plusmn10

3202

103172plusmn10

4326

73205plusmn10

6332

73230plusmn061

188

83203plusmn090

282

3204plusmn090

336

53334plusmn12

0360

63238plusmn074

228

73285plusmn084

255

73286plusmn092

281

3283plusmn096

281

25

3251plusmn

060

185

43390plusmn033

096

43445plusmn15

2442

53362plusmn082

241

3368plusmn12

7377

13338plusmn096

288

43203plusmn026

080

33292plusmn047

143

53277plusmn056

170

3285plusmn079

242

5030

92plusmn047

152

83123plusmn12

9040

83111plusmn13

2041

83108plusmn13

8043

3108plusmn043

138

253177plusmn073

229

83192plusmn14

9048

83191plusmn

093

030

83187plusmn15

7050

3187plusmn051

161

103260plusmn047

144

83281plusmn

061

187

53249plusmn065

199

83263plusmn058

177

3263plusmn057

175

53369plusmn085

253

73382plusmn291

098

53328plusmn250

083

63360plusmn265

089

3357plusmn086

257

25

3436plusmn073

214

53365plusmn18

8063

23327plusmn18

8062

33376plusmn19

6066

3380plusmn080

236

134

55plusmn17

7513

334

97plusmn117

041

53392plusmn341

116

134

48plusmn324

111

3445plusmn13

7397


(b)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CVlowastlowast

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

RotorG

ene1

step

1000

2640plusmn045

169

82374plusmn040

167

82701plusmn

118

438

82572plusmn16

2258

2572plusmn16

2632

500

2749plusmn046

166

82429plusmn034

139

82742plusmn10

4378

82640plusmn16

6228

2640plusmn16

6628

250

2873plusmn076

265

82490plusmn049

196

82927plusmn060

204

72756plusmn209

222

2756plusmn209

758

100

3070plusmn021

067

32550

mdash1

3119plusmn066

211

23000plusmn224

139

3000plusmn224

746

503031

mdash1

mdashmdash

0mdash

mdash0

3031

mdash3031

mdash25

3112plusmn050

161

2mdash

mdash0

mdashmdash

03212plusmn050

161

3212plusmn050

161

5028

87plusmn099

341

829

25plusmn074

252

82797plusmn065

233

828

70plusmn079

276

2870plusmn094

329

2529

49plusmn10

1344

830

36plusmn070

229

828

57plusmn039

136

829

47plusmn070

236

2947plusmn10

3350

1030

29plusmn12

0398

830

61plusmn

144

470

828

76plusmn040

139

829

89plusmn10

1336

2989plusmn13

4450

530

82plusmn10

6343

63147plusmn13

0412

629

90plusmn040

135

830

73plusmn092

297

3065plusmn11

2366

25

3087plusmn114

368

63169plusmn057

181

529

10plusmn037

126

530

55plusmn069

225

3057plusmn13

1429

13122

mdash1

3153

mdash1

3201plusmn

044

137

53159plusmn044

137

3183plusmn048

151

Verso1step

500

2650plusmn018

068

82656plusmn014

053

82679plusmn023

085

82661plusmn

018

069

2661plusmn

022

082

250

2743plusmn018

065

82796plusmn027

098

82789plusmn036

127

82776plusmn027

097

2776plusmn036

130

100

2894plusmn050

172

82942plusmn053

180

82991plusmn

118

396

82943plusmn074

249

2943plusmn087

295

503049plusmn044

143

73138plusmn086

274

83129plusmn15

2487

83105plusmn094

301

3108plusmn10

9350

253282plusmn114

346

73290plusmn052

157

53250plusmn092

282

73274plusmn086

262

3272plusmn090

274

103342plusmn097

291

33522plusmn10

5299

23442

mdash1

3435plusmn10

1295

3419plusmn118

344

250

2939plusmn048

164

829

40plusmn023

079

829

10plusmn053

183

829

20plusmn042

142

2930plusmn044

150

100

3055plusmn025

080

830

32plusmn038

125

830

02plusmn024

082

630

29plusmn029

096

3032plusmn036

119

503116plusmn050

159

83110plusmn052

168

830

87plusmn042

135

73104plusmn048

154

3105plusmn048

153

253171plusmn

032

102

73161plusmn

042

134

83135plusmn058

184

83156plusmn044

140

3155plusmn046

147

103186plusmn054

169

73176plusmn029

091

73165plusmn064

201

43176plusmn049

154

3177plusmn046

145

53309plusmn098

297

53313

13343plusmn074

223

43322plusmn086

260

3326plusmn080

239


(b)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CVlowastlowast

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

qScript1

step

1000

2324plusmn012

053

82297plusmn024

103

82321plusmn

009

037

82314plusmn015

064

2314plusmn020

086

750

2364plusmn021

088

82329plusmn008

035

82352plusmn028

121

82348plusmn019

081

2348plusmn025

106

500

2446plusmn032

132

82406plusmn024

098

82407plusmn029

122

82419plusmn028

117

2419plusmn033

137

250

2538plusmn027

107

82493plusmn026

105

82514plusmn034

137

82515plusmn029

116

2515plusmn034

135

100

2626plusmn031

116

82579plusmn024

093

72595plusmn047

180

52600plusmn034

130

2602plusmn038

146

50mdash

mdash0

mdashmdash

02645plusmn018

070

22645plusmn018

070

2645plusmn018

070

2500

2813plusmn083

294

82778plusmn045

160

826

32plusmn030

113

82741plusmn

052

189

2741plusmn

097

353

1000

2878plusmn009

033

829

40plusmn15

8539

824

89plusmn030

122

82769plusmn066

231

2769plusmn223

805

750

2908plusmn024

082

828

92plusmn025

085

828

75plusmn011

038

828

92plusmn020

068

2892plusmn024

084

500

3069plusmn048

157

729

76plusmn055

186

82763plusmn056

201

529

36plusmn053

181

2966plusmn12

6424

250

3122plusmn023

073

730

05plusmn037

124

8mdash

mdash0

3064plusmn030

099

3060plusmn067

220

100

mdashmdash

030

00

mdash1

mdashmdash

030

00

mdash30

00

mdash

SSIIIP

latinium

1step

1000

2479plusmn024

097

82381plusmn

021

089

82446plusmn015

060

82435plusmn020

082

2435plusmn046

188

750

2518plusmn024

095

82430plusmn012

051

82497plusmn010

042

82482plusmn016

063

2482plusmn041

167

500

2666plusmn082

307

82536plusmn023

092

82619plusmn024

092

82607plusmn043

164

2607plusmn073

282

250

2866plusmn086

301

82838plusmn19

1672

82897plusmn059

205

82867plusmn112

393

2867plusmn12

2427

100

2917plusmn053

180

82778plusmn049

175

82816plusmn053

189

72837plusmn052

182

2838plusmn078

276

503095plusmn043

139

22911plusmn067

231

42993plusmn111

370

33000plusmn074

247

2979plusmn10

3347

250

2627plusmn026

099

826

70plusmn027

102

826

67plusmn042

156

826

55plusmn037

119

2655plusmn037

139

100

2722plusmn068

248

828

02plusmn039

138

82774plusmn034

121

82766plusmn058

169

2766plusmn058

209

502756plusmn053

194

828

26plusmn041

147

828

33plusmn040

141

828

05plusmn056

161

2805plusmn056

200

252787plusmn069

247

829

34plusmn055

189

828

58plusmn041

145

828

59plusmn082

194

2859plusmn082

285

1028

75plusmn078

270

629

44plusmn055

186

529

08plusmn033

114

729

07plusmn060

190

2907plusmn060

207

528

82plusmn015

053

428

71plusmn

021

072

328

88plusmn032

112

428

81plusmn

023

079

2881plusmn

023

079

Theintra-a

ndinterassay

varia

bilityandcoeffi

ciento

fvariatio

nswerec

alculated


=standard

deviationof



100intra-assayCV

=mean

oftheC

Vso

fallruns

lowastAv

erageC


deviation

lowastlowastCV

()=

standard



Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)






RotorGene QMDx

Mx3005P






4 Discussion












5 Conclusion



Abbreviations




Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Table5Analysis

ofreprod

ucibilityof

one-ste

pandtwo-ste

pRT

-PCR

(a)

show

sfor

each

two-ste

pkittheintra-a

ndinterassay

reprod


nsof

EBLV

-1teste

deighttim

esin

each

ofthreeind

ependent

runs


nd(b)sho

wsthe


ucibilityfora

llteste

don

e-ste

pkits

(a)2-ste

pmetho

ds

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CV ()lowastlowast

AverageC

tplusmnSD

CtCV

()lowastlowast

IScriptR

T+qP

CRSsoF

ast

100

3046plusmn041

135

83008plusmn057

190

82982plusmn041

138

83012plusmn046

154

3012plusmn052

174

503111plusmn093

299

63184plusmn10

1319

63080plusmn046

151

83125plusmn080

256

3120plusmn088

283

253249plusmn059

183

63160plusmn067

212

83127plusmn068

219

63179plusmn065

204

3177plusmn080

251

103238plusmn091

281

63269plusmn119

363

63197plusmn076

239

53235plusmn095

294

3237plusmn097

299

53314

mdash1

mdashmdash

03198plusmn005

015

33256plusmn005

015

3227plusmn058

181

05

3217

mdash1

mdashmdash

03211plusmn076

236

33214plusmn076

236

3213plusmn062

193

250

3071plusmn

049

161

830

70plusmn039

128

830

61plusmn

033

108

830

67plusmn041

132

3067plusmn040

129

100

3230plusmn074

228

83203plusmn090

281

83219plusmn040

124

83217plusmn068

211

3217plusmn069

214

5034

02plusmn093

273

63324plusmn048

144

83316plusmn093

287

83347plusmn079

235

3342plusmn085

255

2534

63plusmn10

2295

83378plusmn075

223

634

24plusmn088

258

634

21plusmn

089

259

3425plusmn093

272

103595plusmn11

2312

53526plusmn047

134

534

68plusmn00

63

180

63530plusmn074

209

3526plusmn090

256

53384plusmn062

184

3mdash

mdash0

mdashmdash

034

83plusmn060

171

3384plusmn062

184

QuantiTectR

T+qP

CRQuantitect

250

3137plusmn069

220

83145plusmn052

165

83122plusmn046

147

83135plusmn056

177

3135plusmn055

175

100

3246plusmn080

246

73272plusmn092

281

73390plusmn090

265

73303plusmn087

264

3303plusmn10

5318

503359plusmn060

179

43434plusmn232

675

53366plusmn079

235

63386plusmn12

4363

3387plusmn14

0413

253298plusmn074

224

43358plusmn118

351

53412plusmn207

608

43356plusmn13

3394

3356plusmn13

8410

103354plusmn018

054

33386plusmn061

180

23473plusmn062

179

23404plusmn047

138

3397plusmn065

192

5mdash

mdash0

3552

mdash1

mdashmdash

03552

mdash3552

mdash50

034

09plusmn036

135

836

37plusmn118

326

63297plusmn038

116

834

30plusmn15

419

234

30plusmn15

4448

250

3642plusmn046

643

83366plusmn080

239

83361plusmn

083

246

834

56plusmn19

7376

3456plusmn19

7569

100

3439plusmn234

257

834

60plusmn098

284

834

50plusmn091

265

834

50plusmn089

269

3450plusmn089

258

5036

28plusmn088

475

63507plusmn091

259

734

92plusmn10

4297

73538plusmn13

2344

3538plusmn13

2374

253597plusmn17

2230

536

28plusmn088

242

436

44plusmn078

214

536

23plusmn079

229

3623plusmn079

217

10mdash

mdash0

mdashmdash

03765plusmn079

210

23765plusmn079

210

3765plusmn079

210


(a)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CV ()lowastlowast

AverageC

tplusmnSD

CtCV

()lowastlowast

Maxim

aRT

+qP

CRMaxim

a

100

3183plusmn084

263

3138plusmn022

070

3009plusmn041

137

311plusmn049

157

311plusmn092

295

503335plusmn10

9326

83342plusmn088

264

53112plusmn064

205

83263plusmn087

265

3251plusmn

140

432

253367plusmn088

261

53277plusmn112

340

63247plusmn072

221

73297plusmn090

274

3290plusmn099

302

103716plusmn464

1249

33400plusmn14

1416

43435plusmn13

3388

53517plusmn246

684

3494plusmn263

754

53544plusmn024

068

23865

mdash1

3458plusmn12

4357

33622plusmn074

212

3555plusmn17

6496

25

mdashmdash

03314

mdash1

3347plusmn17

6526

23330plusmn17

6526

3336plusmn12

6377

250

2928plusmn058

199

829

05plusmn038

130

829

04plusmn039

135

829

12plusmn045

155

2912plusmn045

156

100

3053plusmn056

185

830

17plusmn048

159

830

31plusmn

043

143

830

34plusmn050

162

3034plusmn050

164

503154plusmn12

5397

83188plusmn13

7429

83172plusmn056

177

83171plusmn

108

334

3171plusmn

108

340

253300plusmn115

348

63238plusmn079

243

73238plusmn082

254

73256plusmn092

282

3256plusmn092

281

103308plusmn064

193

33302plusmn13

8417

43340plusmn14

6438

43317plusmn115

349

3317plusmn11

5347

503303plusmn015

045

23355

mdash1

3299

mdash1

3315plusmn028

045

3315plusmn028

085

qScriptR

T+qP

CRPerfe

cta

250

2704plusmn024

088

82675plusmn026

098

82677plusmn024

090

82685plusmn025

092

2685plusmn027

101

100

3054plusmn054

176

82984plusmn054

182

83046plusmn10

7350

83028plusmn072

236

3028plusmn079

261

503203plusmn290

905

73072plusmn089

292

83078plusmn082

267

83118plusmn15

4488

3114plusmn17

7568

253250plusmn098

302

73241plusmn

133

410

73363plusmn216

642

53285plusmn14

9452

3276plusmn14

9456

103222plusmn081

252

53287plusmn046

139

33270plusmn056

171

23260plusmn061

187

3251plusmn

069

211

53278plusmn015

045

2mdash

mdash0

3356plusmn15

5461

23318plusmn085

253

3318plusmn10

0303

250

2947plusmn022

074

829

49plusmn054

183

729

82plusmn038

129

829

60plusmn041

129

2960plusmn041

139

100

3099plusmn10

9352

83114plusmn12

6404

83102plusmn052

169

83105plusmn096

308

3105plusmn096

311

503187plusmn060

188

83137plusmn057

180

83313plusmn11

2337

83212plusmn10

8235

3212plusmn10

8335

253335plusmn299

896

73249plusmn068

211

83271plusmn

087

265

83283plusmn17

2457

3283plusmn17

2524

103300plusmn049

147

53345plusmn018

053

23298plusmn074

225

43307plusmn054

142

3307plusmn054

165

53304plusmn10

1306

23303plusmn074

223

23359

mdash1

3314plusmn067

265

3314plusmn067

203


(a)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CV ()lowastlowast

AverageC

tplusmnSD

CtCV

()lowastlowast

SSViloRT

+qP

CREx

press

100

2768plusmn059

213

82782plusmn055

197

82759plusmn029

106

82770plusmn048

172

2770plusmn048

174

502942plusmn062

211

82927plusmn054

185

82921plusmn

060

206

82930plusmn059

201

2930plusmn057

195

253038plusmn067

221

63022plusmn12

4411

82960plusmn048

162

83007plusmn080

264

3004plusmn090

300

103136plusmn049

158

73130plusmn071

227

73192plusmn078

245

63153plusmn066

210

3151plusmn

069

219

53149plusmn053

168

43163plusmn098

308

43164

mdash1

3159plusmn075

238

3157plusmn068

216

05

mdashmdash

0mdash

mdash0

3159

mdash1

3159

mdash3159

mdash100

3178plusmn053

167

830

13plusmn032

107

830

75plusmn065

211

830

89plusmn085

162

3089plusmn085

276

503296plusmn042

129

83134plusmn050

161

83149plusmn097

307

83193plusmn099

199

3193plusmn099

309

253372plusmn10

5310

73215plusmn098

306

83209plusmn022

069

63266plusmn113

228

3266plusmn11

3346

1034

44plusmn060

174

73361plusmn

046

135

53330plusmn043

129

63383plusmn071

146

3383plusmn071

209

53521plusmn

053

151

73264plusmn036

111

53299plusmn006

017

23397plusmn13

5093

3397plusmn13

5398

25

3956plusmn18

6471

334

35

13355plusmn057

169

336

24plusmn332

320

3624plusmn332

916


varia

bilitya

ndcoeffi

ciento

fvariatio

nswerec

alculated


CV=sta



100intra-assayC

V=mean

oftheC

Vso

fallruns

lowastAv

erageC


deviation

lowastlowastCV

()=

standard



Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)

(b)1stepmetho

ds

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CVlowastlowast

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Quantitect1step

502976plusmn064

216

82894plusmn055

189

82927plusmn023

079

82932plusmn047

161

2932plusmn059

106

253090plusmn088

286

82984plusmn10

9365

83098plusmn076

245

83057plusmn091

299

3057plusmn10

3202

103172plusmn10

4326

73205plusmn10

6332

73230plusmn061

188

83203plusmn090

282

3204plusmn090

336

53334plusmn12

0360

63238plusmn074

228

73285plusmn084

255

73286plusmn092

281

3283plusmn096

281

25

3251plusmn

060

185

43390plusmn033

096

43445plusmn15

2442

53362plusmn082

241

3368plusmn12

7377

13338plusmn096

288

43203plusmn026

080

33292plusmn047

143

53277plusmn056

170

3285plusmn079

242

5030

92plusmn047

152

83123plusmn12

9040

83111plusmn13

2041

83108plusmn13

8043

3108plusmn043

138

253177plusmn073

229

83192plusmn14

9048

83191plusmn

093

030

83187plusmn15

7050

3187plusmn051

161

103260plusmn047

144

83281plusmn

061

187

53249plusmn065

199

83263plusmn058

177

3263plusmn057

175

53369plusmn085

253

73382plusmn291

098

53328plusmn250

083

63360plusmn265

089

3357plusmn086

257

25

3436plusmn073

214

53365plusmn18

8063

23327plusmn18

8062

33376plusmn19

6066

3380plusmn080

236

134

55plusmn17

7513

334

97plusmn117

041

53392plusmn341

116

134

48plusmn324

111

3445plusmn13

7397


(b)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CVlowastlowast

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

RotorG

ene1

step

1000

2640plusmn045

169

82374plusmn040

167

82701plusmn

118

438

82572plusmn16

2258

2572plusmn16

2632

500

2749plusmn046

166

82429plusmn034

139

82742plusmn10

4378

82640plusmn16

6228

2640plusmn16

6628

250

2873plusmn076

265

82490plusmn049

196

82927plusmn060

204

72756plusmn209

222

2756plusmn209

758

100

3070plusmn021

067

32550

mdash1

3119plusmn066

211

23000plusmn224

139

3000plusmn224

746

503031

mdash1

mdashmdash

0mdash

mdash0

3031

mdash3031

mdash25

3112plusmn050

161

2mdash

mdash0

mdashmdash

03212plusmn050

161

3212plusmn050

161

5028

87plusmn099

341

829

25plusmn074

252

82797plusmn065

233

828

70plusmn079

276

2870plusmn094

329

2529

49plusmn10

1344

830

36plusmn070

229

828

57plusmn039

136

829

47plusmn070

236

2947plusmn10

3350

1030

29plusmn12

0398

830

61plusmn

144

470

828

76plusmn040

139

829

89plusmn10

1336

2989plusmn13

4450

530

82plusmn10

6343

63147plusmn13

0412

629

90plusmn040

135

830

73plusmn092

297

3065plusmn11

2366

25

3087plusmn114

368

63169plusmn057

181

529

10plusmn037

126

530

55plusmn069

225

3057plusmn13

1429

13122

mdash1

3153

mdash1

3201plusmn

044

137

53159plusmn044

137

3183plusmn048

151

Verso1step

500

2650plusmn018

068

82656plusmn014

053

82679plusmn023

085

82661plusmn

018

069

2661plusmn

022

082

250

2743plusmn018

065

82796plusmn027

098

82789plusmn036

127

82776plusmn027

097

2776plusmn036

130

100

2894plusmn050

172

82942plusmn053

180

82991plusmn

118

396

82943plusmn074

249

2943plusmn087

295

503049plusmn044

143

73138plusmn086

274

83129plusmn15

2487

83105plusmn094

301

3108plusmn10

9350

253282plusmn114

346

73290plusmn052

157

53250plusmn092

282

73274plusmn086

262

3272plusmn090

274

103342plusmn097

291

33522plusmn10

5299

23442

mdash1

3435plusmn10

1295

3419plusmn118

344

250

2939plusmn048

164

829

40plusmn023

079

829

10plusmn053

183

829

20plusmn042

142

2930plusmn044

150

100

3055plusmn025

080

830

32plusmn038

125

830

02plusmn024

082

630

29plusmn029

096

3032plusmn036

119

503116plusmn050

159

83110plusmn052

168

830

87plusmn042

135

73104plusmn048

154

3105plusmn048

153

253171plusmn

032

102

73161plusmn

042

134

83135plusmn058

184

83156plusmn044

140

3155plusmn046

147

103186plusmn054

169

73176plusmn029

091

73165plusmn064

201

43176plusmn049

154

3177plusmn046

145

53309plusmn098

297

53313

13343plusmn074

223

43322plusmn086

260

3326plusmn080

239


(b)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CVlowastlowast

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

qScript1

step

1000

2324plusmn012

053

82297plusmn024

103

82321plusmn

009

037

82314plusmn015

064

2314plusmn020

086

750

2364plusmn021

088

82329plusmn008

035

82352plusmn028

121

82348plusmn019

081

2348plusmn025

106

500

2446plusmn032

132

82406plusmn024

098

82407plusmn029

122

82419plusmn028

117

2419plusmn033

137

250

2538plusmn027

107

82493plusmn026

105

82514plusmn034

137

82515plusmn029

116

2515plusmn034

135

100

2626plusmn031

116

82579plusmn024

093

72595plusmn047

180

52600plusmn034

130

2602plusmn038

146

50mdash

mdash0

mdashmdash

02645plusmn018

070

22645plusmn018

070

2645plusmn018

070

2500

2813plusmn083

294

82778plusmn045

160

826

32plusmn030

113

82741plusmn

052

189

2741plusmn

097

353

1000

2878plusmn009

033

829

40plusmn15

8539

824

89plusmn030

122

82769plusmn066

231

2769plusmn223

805

750

2908plusmn024

082

828

92plusmn025

085

828

75plusmn011

038

828

92plusmn020

068

2892plusmn024

084

500

3069plusmn048

157

729

76plusmn055

186

82763plusmn056

201

529

36plusmn053

181

2966plusmn12

6424

250

3122plusmn023

073

730

05plusmn037

124

8mdash

mdash0

3064plusmn030

099

3060plusmn067

220

100

mdashmdash

030

00

mdash1

mdashmdash

030

00

mdash30

00

mdash

SSIIIP

latinium

1step

1000

2479plusmn024

097

82381plusmn

021

089

82446plusmn015

060

82435plusmn020

082

2435plusmn046

188

750

2518plusmn024

095

82430plusmn012

051

82497plusmn010

042

82482plusmn016

063

2482plusmn041

167

500

2666plusmn082

307

82536plusmn023

092

82619plusmn024

092

82607plusmn043

164

2607plusmn073

282

250

2866plusmn086

301

82838plusmn19

1672

82897plusmn059

205

82867plusmn112

393

2867plusmn12

2427

100

2917plusmn053

180

82778plusmn049

175

82816plusmn053

189

72837plusmn052

182

2838plusmn078

276

503095plusmn043

139

22911plusmn067

231

42993plusmn111

370

33000plusmn074

247

2979plusmn10

3347

250

2627plusmn026

099

826

70plusmn027

102

826

67plusmn042

156

826

55plusmn037

119

2655plusmn037

139

100

2722plusmn068

248

828

02plusmn039

138

82774plusmn034

121

82766plusmn058

169

2766plusmn058

209

502756plusmn053

194

828

26plusmn041

147

828

33plusmn040

141

828

05plusmn056

161

2805plusmn056

200

252787plusmn069

247

829

34plusmn055

189

828

58plusmn041

145

828

59plusmn082

194

2859plusmn082

285

1028

75plusmn078

270

629

44plusmn055

186

529

08plusmn033

114

729

07plusmn060

190

2907plusmn060

207

528

82plusmn015

053

428

71plusmn

021

072

328

88plusmn032

112

428

81plusmn

023

079

2881plusmn

023

079

Theintra-a

ndinterassay

varia

bilityandcoeffi

ciento

fvariatio

nswerec

alculated


=standard

deviationof



100intra-assayCV

=mean

oftheC

Vso

fallruns

lowastAv

erageC


deviation

lowastlowastCV

()=

standard



Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)






RotorGene QMDx

Mx3005P






4 Discussion












5 Conclusion



Abbreviations




Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CV ()lowastlowast

AverageC

tplusmnSD

CtCV

()lowastlowast

Maxim

aRT

+qP

CRMaxim

a

100

3183plusmn084

263

3138plusmn022

070

3009plusmn041

137

311plusmn049

157

311plusmn092

295

503335plusmn10

9326

83342plusmn088

264

53112plusmn064

205

83263plusmn087

265

3251plusmn

140

432

253367plusmn088

261

53277plusmn112

340

63247plusmn072

221

73297plusmn090

274

3290plusmn099

302

103716plusmn464

1249

33400plusmn14

1416

43435plusmn13

3388

53517plusmn246

684

3494plusmn263

754

53544plusmn024

068

23865

mdash1

3458plusmn12

4357

33622plusmn074

212

3555plusmn17

6496

25

mdashmdash

03314

mdash1

3347plusmn17

6526

23330plusmn17

6526

3336plusmn12

6377

250

2928plusmn058

199

829

05plusmn038

130

829

04plusmn039

135

829

12plusmn045

155

2912plusmn045

156

100

3053plusmn056

185

830

17plusmn048

159

830

31plusmn

043

143

830

34plusmn050

162

3034plusmn050

164

503154plusmn12

5397

83188plusmn13

7429

83172plusmn056

177

83171plusmn

108

334

3171plusmn

108

340

253300plusmn115

348

63238plusmn079

243

73238plusmn082

254

73256plusmn092

282

3256plusmn092

281

103308plusmn064

193

33302plusmn13

8417

43340plusmn14

6438

43317plusmn115

349

3317plusmn11

5347

503303plusmn015

045

23355

mdash1

3299

mdash1

3315plusmn028

045

3315plusmn028

085

qScriptR

T+qP

CRPerfe

cta

250

2704plusmn024

088

82675plusmn026

098

82677plusmn024

090

82685plusmn025

092

2685plusmn027

101

100

3054plusmn054

176

82984plusmn054

182

83046plusmn10

7350

83028plusmn072

236

3028plusmn079

261

503203plusmn290

905

73072plusmn089

292

83078plusmn082

267

83118plusmn15

4488

3114plusmn17

7568

253250plusmn098

302

73241plusmn

133

410

73363plusmn216

642

53285plusmn14

9452

3276plusmn14

9456

103222plusmn081

252

53287plusmn046

139

33270plusmn056

171

23260plusmn061

187

3251plusmn

069

211

53278plusmn015

045

2mdash

mdash0

3356plusmn15

5461

23318plusmn085

253

3318plusmn10

0303

250

2947plusmn022

074

829

49plusmn054

183

729

82plusmn038

129

829

60plusmn041

129

2960plusmn041

139

100

3099plusmn10

9352

83114plusmn12

6404

83102plusmn052

169

83105plusmn096

308

3105plusmn096

311

503187plusmn060

188

83137plusmn057

180

83313plusmn11

2337

83212plusmn10

8235

3212plusmn10

8335

253335plusmn299

896

73249plusmn068

211

83271plusmn

087

265

83283plusmn17

2457

3283plusmn17

2524

103300plusmn049

147

53345plusmn018

053

23298plusmn074

225

43307plusmn054

142

3307plusmn054

165

53304plusmn10

1306

23303plusmn074

223

23359

mdash1

3314plusmn067

265

3314plusmn067

203


(a)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CV ()lowastlowast

AverageC

tplusmnSD

CtCV

()lowastlowast

SSViloRT

+qP

CREx

press

100

2768plusmn059

213

82782plusmn055

197

82759plusmn029

106

82770plusmn048

172

2770plusmn048

174

502942plusmn062

211

82927plusmn054

185

82921plusmn

060

206

82930plusmn059

201

2930plusmn057

195

253038plusmn067

221

63022plusmn12

4411

82960plusmn048

162

83007plusmn080

264

3004plusmn090

300

103136plusmn049

158

73130plusmn071

227

73192plusmn078

245

63153plusmn066

210

3151plusmn

069

219

53149plusmn053

168

43163plusmn098

308

43164

mdash1

3159plusmn075

238

3157plusmn068

216

05

mdashmdash

0mdash

mdash0

3159

mdash1

3159

mdash3159

mdash100

3178plusmn053

167

830

13plusmn032

107

830

75plusmn065

211

830

89plusmn085

162

3089plusmn085

276

503296plusmn042

129

83134plusmn050

161

83149plusmn097

307

83193plusmn099

199

3193plusmn099

309

253372plusmn10

5310

73215plusmn098

306

83209plusmn022

069

63266plusmn113

228

3266plusmn11

3346

1034

44plusmn060

174

73361plusmn

046

135

53330plusmn043

129

63383plusmn071

146

3383plusmn071

209

53521plusmn

053

151

73264plusmn036

111

53299plusmn006

017

23397plusmn13

5093

3397plusmn13

5398

25

3956plusmn18

6471

334

35

13355plusmn057

169

336

24plusmn332

320

3624plusmn332

916


varia

bilitya

ndcoeffi

ciento

fvariatio

nswerec

alculated


CV=sta



100intra-assayC

V=mean

oftheC

Vso

fallruns

lowastAv

erageC


deviation

lowastlowastCV

()=

standard



Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)

(b)1stepmetho

ds

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CVlowastlowast

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Quantitect1step

502976plusmn064

216

82894plusmn055

189

82927plusmn023

079

82932plusmn047

161

2932plusmn059

106

253090plusmn088

286

82984plusmn10

9365

83098plusmn076

245

83057plusmn091

299

3057plusmn10

3202

103172plusmn10

4326

73205plusmn10

6332

73230plusmn061

188

83203plusmn090

282

3204plusmn090

336

53334plusmn12

0360

63238plusmn074

228

73285plusmn084

255

73286plusmn092

281

3283plusmn096

281

25

3251plusmn

060

185

43390plusmn033

096

43445plusmn15

2442

53362plusmn082

241

3368plusmn12

7377

13338plusmn096

288

43203plusmn026

080

33292plusmn047

143

53277plusmn056

170

3285plusmn079

242

5030

92plusmn047

152

83123plusmn12

9040

83111plusmn13

2041

83108plusmn13

8043

3108plusmn043

138

253177plusmn073

229

83192plusmn14

9048

83191plusmn

093

030

83187plusmn15

7050

3187plusmn051

161

103260plusmn047

144

83281plusmn

061

187

53249plusmn065

199

83263plusmn058

177

3263plusmn057

175

53369plusmn085

253

73382plusmn291

098

53328plusmn250

083

63360plusmn265

089

3357plusmn086

257

25

3436plusmn073

214

53365plusmn18

8063

23327plusmn18

8062

33376plusmn19

6066

3380plusmn080

236

134

55plusmn17

7513

334

97plusmn117

041

53392plusmn341

116

134

48plusmn324

111

3445plusmn13

7397


(b)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CVlowastlowast

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

RotorG

ene1

step

1000

2640plusmn045

169

82374plusmn040

167

82701plusmn

118

438

82572plusmn16

2258

2572plusmn16

2632

500

2749plusmn046

166

82429plusmn034

139

82742plusmn10

4378

82640plusmn16

6228

2640plusmn16

6628

250

2873plusmn076

265

82490plusmn049

196

82927plusmn060

204

72756plusmn209

222

2756plusmn209

758

100

3070plusmn021

067

32550

mdash1

3119plusmn066

211

23000plusmn224

139

3000plusmn224

746

503031

mdash1

mdashmdash

0mdash

mdash0

3031

mdash3031

mdash25

3112plusmn050

161

2mdash

mdash0

mdashmdash

03212plusmn050

161

3212plusmn050

161

5028

87plusmn099

341

829

25plusmn074

252

82797plusmn065

233

828

70plusmn079

276

2870plusmn094

329

2529

49plusmn10

1344

830

36plusmn070

229

828

57plusmn039

136

829

47plusmn070

236

2947plusmn10

3350

1030

29plusmn12

0398

830

61plusmn

144

470

828

76plusmn040

139

829

89plusmn10

1336

2989plusmn13

4450

530

82plusmn10

6343

63147plusmn13

0412

629

90plusmn040

135

830

73plusmn092

297

3065plusmn11

2366

25

3087plusmn114

368

63169plusmn057

181

529

10plusmn037

126

530

55plusmn069

225

3057plusmn13

1429

13122

mdash1

3153

mdash1

3201plusmn

044

137

53159plusmn044

137

3183plusmn048

151

Verso1step

500

2650plusmn018

068

82656plusmn014

053

82679plusmn023

085

82661plusmn

018

069

2661plusmn

022

082

250

2743plusmn018

065

82796plusmn027

098

82789plusmn036

127

82776plusmn027

097

2776plusmn036

130

100

2894plusmn050

172

82942plusmn053

180

82991plusmn

118

396

82943plusmn074

249

2943plusmn087

295

503049plusmn044

143

73138plusmn086

274

83129plusmn15

2487

83105plusmn094

301

3108plusmn10

9350

253282plusmn114

346

73290plusmn052

157

53250plusmn092

282

73274plusmn086

262

3272plusmn090

274

103342plusmn097

291

33522plusmn10

5299

23442

mdash1

3435plusmn10

1295

3419plusmn118

344

250

2939plusmn048

164

829

40plusmn023

079

829

10plusmn053

183

829

20plusmn042

142

2930plusmn044

150

100

3055plusmn025

080

830

32plusmn038

125

830

02plusmn024

082

630

29plusmn029

096

3032plusmn036

119

503116plusmn050

159

83110plusmn052

168

830

87plusmn042

135

73104plusmn048

154

3105plusmn048

153

253171plusmn

032

102

73161plusmn

042

134

83135plusmn058

184

83156plusmn044

140

3155plusmn046

147

103186plusmn054

169

73176plusmn029

091

73165plusmn064

201

43176plusmn049

154

3177plusmn046

145

53309plusmn098

297

53313

13343plusmn074

223

43322plusmn086

260

3326plusmn080

239


(b)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CVlowastlowast

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

qScript1

step

1000

2324plusmn012

053

82297plusmn024

103

82321plusmn

009

037

82314plusmn015

064

2314plusmn020

086

750

2364plusmn021

088

82329plusmn008

035

82352plusmn028

121

82348plusmn019

081

2348plusmn025

106

500

2446plusmn032

132

82406plusmn024

098

82407plusmn029

122

82419plusmn028

117

2419plusmn033

137

250

2538plusmn027

107

82493plusmn026

105

82514plusmn034

137

82515plusmn029

116

2515plusmn034

135

100

2626plusmn031

116

82579plusmn024

093

72595plusmn047

180

52600plusmn034

130

2602plusmn038

146

50mdash

mdash0

mdashmdash

02645plusmn018

070

22645plusmn018

070

2645plusmn018

070

2500

2813plusmn083

294

82778plusmn045

160

826

32plusmn030

113

82741plusmn

052

189

2741plusmn

097

353

1000

2878plusmn009

033

829

40plusmn15

8539

824

89plusmn030

122

82769plusmn066

231

2769plusmn223

805

750

2908plusmn024

082

828

92plusmn025

085

828

75plusmn011

038

828

92plusmn020

068

2892plusmn024

084

500

3069plusmn048

157

729

76plusmn055

186

82763plusmn056

201

529

36plusmn053

181

2966plusmn12

6424

250

3122plusmn023

073

730

05plusmn037

124

8mdash

mdash0

3064plusmn030

099

3060plusmn067

220

100

mdashmdash

030

00

mdash1

mdashmdash

030

00

mdash30

00

mdash

SSIIIP

latinium

1step

1000

2479plusmn024

097

82381plusmn

021

089

82446plusmn015

060

82435plusmn020

082

2435plusmn046

188

750

2518plusmn024

095

82430plusmn012

051

82497plusmn010

042

82482plusmn016

063

2482plusmn041

167

500

2666plusmn082

307

82536plusmn023

092

82619plusmn024

092

82607plusmn043

164

2607plusmn073

282

250

2866plusmn086

301

82838plusmn19

1672

82897plusmn059

205

82867plusmn112

393

2867plusmn12

2427

100

2917plusmn053

180

82778plusmn049

175

82816plusmn053

189

72837plusmn052

182

2838plusmn078

276

503095plusmn043

139

22911plusmn067

231

42993plusmn111

370

33000plusmn074

247

2979plusmn10

3347

250

2627plusmn026

099

826

70plusmn027

102

826

67plusmn042

156

826

55plusmn037

119

2655plusmn037

139

100

2722plusmn068

248

828

02plusmn039

138

82774plusmn034

121

82766plusmn058

169

2766plusmn058

209

502756plusmn053

194

828

26plusmn041

147

828

33plusmn040

141

828

05plusmn056

161

2805plusmn056

200

252787plusmn069

247

829

34plusmn055

189

828

58plusmn041

145

828

59plusmn082

194

2859plusmn082

285

1028

75plusmn078

270

629

44plusmn055

186

529

08plusmn033

114

729

07plusmn060

190

2907plusmn060

207

528

82plusmn015

053

428

71plusmn

021

072

328

88plusmn032

112

428

81plusmn

023

079

2881plusmn

023

079

Theintra-a

ndinterassay

varia

bilityandcoeffi

ciento

fvariatio

nswerec

alculated


=standard

deviationof



100intra-assayCV

=mean

oftheC

Vso

fallruns

lowastAv

erageC


deviation

lowastlowastCV

()=

standard



Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)






RotorGene QMDx

Mx3005P






4 Discussion












5 Conclusion



Abbreviations




Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(a)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CV ()lowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CV ()lowastlowast

AverageC

tplusmnSD

CtCV

()lowastlowast

SSViloRT

+qP

CREx

press

100

2768plusmn059

213

82782plusmn055

197

82759plusmn029

106

82770plusmn048

172

2770plusmn048

174

502942plusmn062

211

82927plusmn054

185

82921plusmn

060

206

82930plusmn059

201

2930plusmn057

195

253038plusmn067

221

63022plusmn12

4411

82960plusmn048

162

83007plusmn080

264

3004plusmn090

300

103136plusmn049

158

73130plusmn071

227

73192plusmn078

245

63153plusmn066

210

3151plusmn

069

219

53149plusmn053

168

43163plusmn098

308

43164

mdash1

3159plusmn075

238

3157plusmn068

216

05

mdashmdash

0mdash

mdash0

3159

mdash1

3159

mdash3159

mdash100

3178plusmn053

167

830

13plusmn032

107

830

75plusmn065

211

830

89plusmn085

162

3089plusmn085

276

503296plusmn042

129

83134plusmn050

161

83149plusmn097

307

83193plusmn099

199

3193plusmn099

309

253372plusmn10

5310

73215plusmn098

306

83209plusmn022

069

63266plusmn113

228

3266plusmn11

3346

1034

44plusmn060

174

73361plusmn

046

135

53330plusmn043

129

63383plusmn071

146

3383plusmn071

209

53521plusmn

053

151

73264plusmn036

111

53299plusmn006

017

23397plusmn13

5093

3397plusmn13

5398

25

3956plusmn18

6471

334

35

13355plusmn057

169

336

24plusmn332

320

3624plusmn332

916


varia

bilitya

ndcoeffi

ciento

fvariatio

nswerec

alculated


CV=sta



100intra-assayC

V=mean

oftheC

Vso

fallruns

lowastAv

erageC


deviation

lowastlowastCV

()=

standard



Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)

(b)1stepmetho

ds

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CVlowastlowast

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Quantitect1step

502976plusmn064

216

82894plusmn055

189

82927plusmn023

079

82932plusmn047

161

2932plusmn059

106

253090plusmn088

286

82984plusmn10

9365

83098plusmn076

245

83057plusmn091

299

3057plusmn10

3202

103172plusmn10

4326

73205plusmn10

6332

73230plusmn061

188

83203plusmn090

282

3204plusmn090

336

53334plusmn12

0360

63238plusmn074

228

73285plusmn084

255

73286plusmn092

281

3283plusmn096

281

25

3251plusmn

060

185

43390plusmn033

096

43445plusmn15

2442

53362plusmn082

241

3368plusmn12

7377

13338plusmn096

288

43203plusmn026

080

33292plusmn047

143

53277plusmn056

170

3285plusmn079

242

5030

92plusmn047

152

83123plusmn12

9040

83111plusmn13

2041

83108plusmn13

8043

3108plusmn043

138

253177plusmn073

229

83192plusmn14

9048

83191plusmn

093

030

83187plusmn15

7050

3187plusmn051

161

103260plusmn047

144

83281plusmn

061

187

53249plusmn065

199

83263plusmn058

177

3263plusmn057

175

53369plusmn085

253

73382plusmn291

098

53328plusmn250

083

63360plusmn265

089

3357plusmn086

257

25

3436plusmn073

214

53365plusmn18

8063

23327plusmn18

8062

33376plusmn19

6066

3380plusmn080

236

134

55plusmn17

7513

334

97plusmn117

041

53392plusmn341

116

134

48plusmn324

111

3445plusmn13

7397


(b)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CVlowastlowast

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

RotorG

ene1

step

1000

2640plusmn045

169

82374plusmn040

167

82701plusmn

118

438

82572plusmn16

2258

2572plusmn16

2632

500

2749plusmn046

166

82429plusmn034

139

82742plusmn10

4378

82640plusmn16

6228

2640plusmn16

6628

250

2873plusmn076

265

82490plusmn049

196

82927plusmn060

204

72756plusmn209

222

2756plusmn209

758

100

3070plusmn021

067

32550

mdash1

3119plusmn066

211

23000plusmn224

139

3000plusmn224

746

503031

mdash1

mdashmdash

0mdash

mdash0

3031

mdash3031

mdash25

3112plusmn050

161

2mdash

mdash0

mdashmdash

03212plusmn050

161

3212plusmn050

161

5028

87plusmn099

341

829

25plusmn074

252

82797plusmn065

233

828

70plusmn079

276

2870plusmn094

329

2529

49plusmn10

1344

830

36plusmn070

229

828

57plusmn039

136

829

47plusmn070

236

2947plusmn10

3350

1030

29plusmn12

0398

830

61plusmn

144

470

828

76plusmn040

139

829

89plusmn10

1336

2989plusmn13

4450

530

82plusmn10

6343

63147plusmn13

0412

629

90plusmn040

135

830

73plusmn092

297

3065plusmn11

2366

25

3087plusmn114

368

63169plusmn057

181

529

10plusmn037

126

530

55plusmn069

225

3057plusmn13

1429

13122

mdash1

3153

mdash1

3201plusmn

044

137

53159plusmn044

137

3183plusmn048

151

Verso1step

500

2650plusmn018

068

82656plusmn014

053

82679plusmn023

085

82661plusmn

018

069

2661plusmn

022

082

250

2743plusmn018

065

82796plusmn027

098

82789plusmn036

127

82776plusmn027

097

2776plusmn036

130

100

2894plusmn050

172

82942plusmn053

180

82991plusmn

118

396

82943plusmn074

249

2943plusmn087

295

503049plusmn044

143

73138plusmn086

274

83129plusmn15

2487

83105plusmn094

301

3108plusmn10

9350

253282plusmn114

346

73290plusmn052

157

53250plusmn092

282

73274plusmn086

262

3272plusmn090

274

103342plusmn097

291

33522plusmn10

5299

23442

mdash1

3435plusmn10

1295

3419plusmn118

344

250

2939plusmn048

164

829

40plusmn023

079

829

10plusmn053

183

829

20plusmn042

142

2930plusmn044

150

100

3055plusmn025

080

830

32plusmn038

125

830

02plusmn024

082

630

29plusmn029

096

3032plusmn036

119

503116plusmn050

159

83110plusmn052

168

830

87plusmn042

135

73104plusmn048

154

3105plusmn048

153

253171plusmn

032

102

73161plusmn

042

134

83135plusmn058

184

83156plusmn044

140

3155plusmn046

147

103186plusmn054

169

73176plusmn029

091

73165plusmn064

201

43176plusmn049

154

3177plusmn046

145

53309plusmn098

297

53313

13343plusmn074

223

43322plusmn086

260

3326plusmn080

239


(b)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CVlowastlowast

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

qScript1

step

1000

2324plusmn012

053

82297plusmn024

103

82321plusmn

009

037

82314plusmn015

064

2314plusmn020

086

750

2364plusmn021

088

82329plusmn008

035

82352plusmn028

121

82348plusmn019

081

2348plusmn025

106

500

2446plusmn032

132

82406plusmn024

098

82407plusmn029

122

82419plusmn028

117

2419plusmn033

137

250

2538plusmn027

107

82493plusmn026

105

82514plusmn034

137

82515plusmn029

116

2515plusmn034

135

100

2626plusmn031

116

82579plusmn024

093

72595plusmn047

180

52600plusmn034

130

2602plusmn038

146

50mdash

mdash0

mdashmdash

02645plusmn018

070

22645plusmn018

070

2645plusmn018

070

2500

2813plusmn083

294

82778plusmn045

160

826

32plusmn030

113

82741plusmn

052

189

2741plusmn

097

353

1000

2878plusmn009

033

829

40plusmn15

8539

824

89plusmn030

122

82769plusmn066

231

2769plusmn223

805

750

2908plusmn024

082

828

92plusmn025

085

828

75plusmn011

038

828

92plusmn020

068

2892plusmn024

084

500

3069plusmn048

157

729

76plusmn055

186

82763plusmn056

201

529

36plusmn053

181

2966plusmn12

6424

250

3122plusmn023

073

730

05plusmn037

124

8mdash

mdash0

3064plusmn030

099

3060plusmn067

220

100

mdashmdash

030

00

mdash1

mdashmdash

030

00

mdash30

00

mdash

SSIIIP

latinium

1step

1000

2479plusmn024

097

82381plusmn

021

089

82446plusmn015

060

82435plusmn020

082

2435plusmn046

188

750

2518plusmn024

095

82430plusmn012

051

82497plusmn010

042

82482plusmn016

063

2482plusmn041

167

500

2666plusmn082

307

82536plusmn023

092

82619plusmn024

092

82607plusmn043

164

2607plusmn073

282

250

2866plusmn086

301

82838plusmn19

1672

82897plusmn059

205

82867plusmn112

393

2867plusmn12

2427

100

2917plusmn053

180

82778plusmn049

175

82816plusmn053

189

72837plusmn052

182

2838plusmn078

276

503095plusmn043

139

22911plusmn067

231

42993plusmn111

370

33000plusmn074

247

2979plusmn10

3347

250

2627plusmn026

099

826

70plusmn027

102

826

67plusmn042

156

826

55plusmn037

119

2655plusmn037

139

100

2722plusmn068

248

828

02plusmn039

138

82774plusmn034

121

82766plusmn058

169

2766plusmn058

209

502756plusmn053

194

828

26plusmn041

147

828

33plusmn040

141

828

05plusmn056

161

2805plusmn056

200

252787plusmn069

247

829

34plusmn055

189

828

58plusmn041

145

828

59plusmn082

194

2859plusmn082

285

1028

75plusmn078

270

629

44plusmn055

186

529

08plusmn033

114

729

07plusmn060

190

2907plusmn060

207

528

82plusmn015

053

428

71plusmn

021

072

328

88plusmn032

112

428

81plusmn

023

079

2881plusmn

023

079

Theintra-a

ndinterassay

varia

bilityandcoeffi

ciento

fvariatio

nswerec

alculated


=standard

deviationof



100intra-assayCV

=mean

oftheC

Vso

fallruns

lowastAv

erageC


deviation

lowastlowastCV

()=

standard



Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)






RotorGene QMDx

Mx3005P






4 Discussion












5 Conclusion



Abbreviations




Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(b)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CVlowastlowast

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

RotorG

ene1

step

1000

2640plusmn045

169

82374plusmn040

167

82701plusmn

118

438

82572plusmn16

2258

2572plusmn16

2632

500

2749plusmn046

166

82429plusmn034

139

82742plusmn10

4378

82640plusmn16

6228

2640plusmn16

6628

250

2873plusmn076

265

82490plusmn049

196

82927plusmn060

204

72756plusmn209

222

2756plusmn209

758

100

3070plusmn021

067

32550

mdash1

3119plusmn066

211

23000plusmn224

139

3000plusmn224

746

503031

mdash1

mdashmdash

0mdash

mdash0

3031

mdash3031

mdash25

3112plusmn050

161

2mdash

mdash0

mdashmdash

03212plusmn050

161

3212plusmn050

161

5028

87plusmn099

341

829

25plusmn074

252

82797plusmn065

233

828

70plusmn079

276

2870plusmn094

329

2529

49plusmn10

1344

830

36plusmn070

229

828

57plusmn039

136

829

47plusmn070

236

2947plusmn10

3350

1030

29plusmn12

0398

830

61plusmn

144

470

828

76plusmn040

139

829

89plusmn10

1336

2989plusmn13

4450

530

82plusmn10

6343

63147plusmn13

0412

629

90plusmn040

135

830

73plusmn092

297

3065plusmn11

2366

25

3087plusmn114

368

63169plusmn057

181

529

10plusmn037

126

530

55plusmn069

225

3057plusmn13

1429

13122

mdash1

3153

mdash1

3201plusmn

044

137

53159plusmn044

137

3183plusmn048

151

Verso1step

500

2650plusmn018

068

82656plusmn014

053

82679plusmn023

085

82661plusmn

018

069

2661plusmn

022

082

250

2743plusmn018

065

82796plusmn027

098

82789plusmn036

127

82776plusmn027

097

2776plusmn036

130

100

2894plusmn050

172

82942plusmn053

180

82991plusmn

118

396

82943plusmn074

249

2943plusmn087

295

503049plusmn044

143

73138plusmn086

274

83129plusmn15

2487

83105plusmn094

301

3108plusmn10

9350

253282plusmn114

346

73290plusmn052

157

53250plusmn092

282

73274plusmn086

262

3272plusmn090

274

103342plusmn097

291

33522plusmn10

5299

23442

mdash1

3435plusmn10

1295

3419plusmn118

344

250

2939plusmn048

164

829

40plusmn023

079

829

10plusmn053

183

829

20plusmn042

142

2930plusmn044

150

100

3055plusmn025

080

830

32plusmn038

125

830

02plusmn024

082

630

29plusmn029

096

3032plusmn036

119

503116plusmn050

159

83110plusmn052

168

830

87plusmn042

135

73104plusmn048

154

3105plusmn048

153

253171plusmn

032

102

73161plusmn

042

134

83135plusmn058

184

83156plusmn044

140

3155plusmn046

147

103186plusmn054

169

73176plusmn029

091

73165plusmn064

201

43176plusmn049

154

3177plusmn046

145

53309plusmn098

297

53313

13343plusmn074

223

43322plusmn086

260

3326plusmn080

239


(b)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CVlowastlowast

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

qScript1

step

1000

2324plusmn012

053

82297plusmn024

103

82321plusmn

009

037

82314plusmn015

064

2314plusmn020

086

750

2364plusmn021

088

82329plusmn008

035

82352plusmn028

121

82348plusmn019

081

2348plusmn025

106

500

2446plusmn032

132

82406plusmn024

098

82407plusmn029

122

82419plusmn028

117

2419plusmn033

137

250

2538plusmn027

107

82493plusmn026

105

82514plusmn034

137

82515plusmn029

116

2515plusmn034

135

100

2626plusmn031

116

82579plusmn024

093

72595plusmn047

180

52600plusmn034

130

2602plusmn038

146

50mdash

mdash0

mdashmdash

02645plusmn018

070

22645plusmn018

070

2645plusmn018

070

2500

2813plusmn083

294

82778plusmn045

160

826

32plusmn030

113

82741plusmn

052

189

2741plusmn

097

353

1000

2878plusmn009

033

829

40plusmn15

8539

824

89plusmn030

122

82769plusmn066

231

2769plusmn223

805

750

2908plusmn024

082

828

92plusmn025

085

828

75plusmn011

038

828

92plusmn020

068

2892plusmn024

084

500

3069plusmn048

157

729

76plusmn055

186

82763plusmn056

201

529

36plusmn053

181

2966plusmn12

6424

250

3122plusmn023

073

730

05plusmn037

124

8mdash

mdash0

3064plusmn030

099

3060plusmn067

220

100

mdashmdash

030

00

mdash1

mdashmdash

030

00

mdash30

00

mdash

SSIIIP

latinium

1step

1000

2479plusmn024

097

82381plusmn

021

089

82446plusmn015

060

82435plusmn020

082

2435plusmn046

188

750

2518plusmn024

095

82430plusmn012

051

82497plusmn010

042

82482plusmn016

063

2482plusmn041

167

500

2666plusmn082

307

82536plusmn023

092

82619plusmn024

092

82607plusmn043

164

2607plusmn073

282

250

2866plusmn086

301

82838plusmn19

1672

82897plusmn059

205

82867plusmn112

393

2867plusmn12

2427

100

2917plusmn053

180

82778plusmn049

175

82816plusmn053

189

72837plusmn052

182

2838plusmn078

276

503095plusmn043

139

22911plusmn067

231

42993plusmn111

370

33000plusmn074

247

2979plusmn10

3347

250

2627plusmn026

099

826

70plusmn027

102

826

67plusmn042

156

826

55plusmn037

119

2655plusmn037

139

100

2722plusmn068

248

828

02plusmn039

138

82774plusmn034

121

82766plusmn058

169

2766plusmn058

209

502756plusmn053

194

828

26plusmn041

147

828

33plusmn040

141

828

05plusmn056

161

2805plusmn056

200

252787plusmn069

247

829

34plusmn055

189

828

58plusmn041

145

828

59plusmn082

194

2859plusmn082

285

1028

75plusmn078

270

629

44plusmn055

186

529

08plusmn033

114

729

07plusmn060

190

2907plusmn060

207

528

82plusmn015

053

428

71plusmn

021

072

328

88plusmn032

112

428

81plusmn

023

079

2881plusmn

023

079

Theintra-a

ndinterassay

varia

bilityandcoeffi

ciento

fvariatio

nswerec

alculated


=standard

deviationof



100intra-assayCV

=mean

oftheC

Vso

fallruns

lowastAv

erageC


deviation

lowastlowastCV

()=

standard



Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)






RotorGene QMDx

Mx3005P






4 Discussion












5 Conclusion



Abbreviations




Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


(b)Con

tinued

Kit

Num

bero

fcopiessam

ple

Intra-assayvaria


Inter-assayvaria


Run1

Run2

Run3

Average

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sAv

erageC

tplusmnSD

Ctlowast

CVlowastlowast

Num

ber

ofpo

sCtplusmnSD

Ctlowast

CVlowastlowast

AverageC

tplusmnSD

Ctlowast

CVlowastlowast

qScript1

step

1000

2324plusmn012

053

82297plusmn024

103

82321plusmn

009

037

82314plusmn015

064

2314plusmn020

086

750

2364plusmn021

088

82329plusmn008

035

82352plusmn028

121

82348plusmn019

081

2348plusmn025

106

500

2446plusmn032

132

82406plusmn024

098

82407plusmn029

122

82419plusmn028

117

2419plusmn033

137

250

2538plusmn027

107

82493plusmn026

105

82514plusmn034

137

82515plusmn029

116

2515plusmn034

135

100

2626plusmn031

116

82579plusmn024

093

72595plusmn047

180

52600plusmn034

130

2602plusmn038

146

50mdash

mdash0

mdashmdash

02645plusmn018

070

22645plusmn018

070

2645plusmn018

070

2500

2813plusmn083

294

82778plusmn045

160

826

32plusmn030

113

82741plusmn

052

189

2741plusmn

097

353

1000

2878plusmn009

033

829

40plusmn15

8539

824

89plusmn030

122

82769plusmn066

231

2769plusmn223

805

750

2908plusmn024

082

828

92plusmn025

085

828

75plusmn011

038

828

92plusmn020

068

2892plusmn024

084

500

3069plusmn048

157

729

76plusmn055

186

82763plusmn056

201

529

36plusmn053

181

2966plusmn12

6424

250

3122plusmn023

073

730

05plusmn037

124

8mdash

mdash0

3064plusmn030

099

3060plusmn067

220

100

mdashmdash

030

00

mdash1

mdashmdash

030

00

mdash30

00

mdash

SSIIIP

latinium

1step

1000

2479plusmn024

097

82381plusmn

021

089

82446plusmn015

060

82435plusmn020

082

2435plusmn046

188

750

2518plusmn024

095

82430plusmn012

051

82497plusmn010

042

82482plusmn016

063

2482plusmn041

167

500

2666plusmn082

307

82536plusmn023

092

82619plusmn024

092

82607plusmn043

164

2607plusmn073

282

250

2866plusmn086

301

82838plusmn19

1672

82897plusmn059

205

82867plusmn112

393

2867plusmn12

2427

100

2917plusmn053

180

82778plusmn049

175

82816plusmn053

189

72837plusmn052

182

2838plusmn078

276

503095plusmn043

139

22911plusmn067

231

42993plusmn111

370

33000plusmn074

247

2979plusmn10

3347

250

2627plusmn026

099

826

70plusmn027

102

826

67plusmn042

156

826

55plusmn037

119

2655plusmn037

139

100

2722plusmn068

248

828

02plusmn039

138

82774plusmn034

121

82766plusmn058

169

2766plusmn058

209

502756plusmn053

194

828

26plusmn041

147

828

33plusmn040

141

828

05plusmn056

161

2805plusmn056

200

252787plusmn069

247

829

34plusmn055

189

828

58plusmn041

145

828

59plusmn082

194

2859plusmn082

285

1028

75plusmn078

270

629

44plusmn055

186

529

08plusmn033

114

729

07plusmn060

190

2907plusmn060

207

528

82plusmn015

053

428

71plusmn

021

072

328

88plusmn032

112

428

81plusmn

023

079

2881plusmn

023

079

Theintra-a

ndinterassay

varia

bilityandcoeffi

ciento

fvariatio

nswerec

alculated


=standard

deviationof



100intra-assayCV

=mean

oftheC

Vso

fallruns

lowastAv

erageC


deviation

lowastlowastCV

()=

standard



Rcomparis

onsw

erer

unon

theR

otor

GeneQ

MDxthermocycler(in

italic

font)a

ndon

theM

x3005P

thermocycler(in

bold)






RotorGene QMDx

Mx3005P






4 Discussion












5 Conclusion



Abbreviations




Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






RotorGene QMDx

Mx3005P






4 Discussion












5 Conclusion



Abbreviations




Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







5 Conclusion



Abbreviations




Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


Abbreviations




Acknowledgments


References















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article cross-platform evaluation of commercial real-time sybr green rt-pcr...

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