research article a taqman real-time pcr assay for...

Hindawi Publishing CorporationThe Scientific World JournalVolume 2013 Article ID 942682 9 pageshttpdxdoiorg1011552013942682

Research ArticleA TaqMan Real-Time PCR Assay for Detection andQuantification of Sporisorium scitamineum in Sugarcane

Yachun Su Shanshan Wang Jinlong Guo Bantong Xue Liping Xu and Youxiong Que

Key Laboratory of Sugarcane Biology and Genetic Breeding Ministry of AgricultureFujian Agriculture and Forestry UniversityFuzhou 350002 China

Correspondence should be addressed to Liping Xu xlpmail126com and Youxiong Que queyouxionghotmailcom

Received 1 August 2013 Accepted 4 September 2013

Academic Editors V Cattori J C Marshall and B Tian

Copyright copy 2013 Yachun Su et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Sporisorium scitamineum is a fungal smut pathogen epidemic in sugarcane producing areas Early detection and properidentification of the smut are an essential requirement in its management practice In this study we developed a TaqManreal-time PCR assay using specific primers (bEQ-FbEQ-R) and a TaqMan probe (bEQ-P) which were designed based onthe bE (b East mating type) gene (Genbank Accession no U612901) This method was more sensitive (a detection limit of10 ag pbE DNA and 08 ng sugarcane genomic DNA) than that of conventional PCR (10 fg and 100 ng resp) Reliabilitywas demonstrated through the positive detection of samples collected from artificially inoculated sugarcane plantlets (FN40)This assay was capable of detecting the smut pathogen at the initial stage (12 h) of infection and suitable for inspection ofsugarcane pathogen-free seed cane and seedlings Furthermore quantification of pathogen was verified in pathogen-challengedbuds in different sugarcane genotypes which suggested its feasibility for evaluation of smut resistance in different sugarcanegenotypes Taken together this novel assay can be used as a diagnostic tool for sensitive accurate fast and quantitativedetection of the smut pathogen especially for asymptomatic seed cane or plants and evaluation of smut resistance of sugarcanegenotypes

1 Introduction

Sugarcane smut (Sporisorium scitamineum) is a diseaseworldwide and presents in almost all sugarcane-plantingcountries [1 2] The typical feature of sugarcane infectedwith the smut is the emergence of black whips around 120days of planting [3] It is impossible to completely eliminatethis disease once the smut appears due to the enormousquantity of teliospores and the quick spread within the sug-arcane growing area The infected buds may either producesymptoms or exist as a latent infection which may germinateand produce black whips in the following season [4] Thesmut spores germinate on the internal surface followed byformation of appressoria on the inner scales of the youngbuds as well as the base of the emerging leaves Entry into themeristem of the buds occurs between 6 h and 36 h after theteliospore deposition [5] The disease commonly producesplants with profuse tillering narrow leaves spindly shoots

and poor cane therefore causing serious losses in cane yieldand sugar yield per unit area

The development and severity of the smut disease dependon environmental conditions the resistance of sugarcanegenotypes and the interaction between sugarcane S sporiso-rium and environment Previous researches revealed thatthe control of sugarcane smut is difficult and the successfulmanagement relies mainly on the use of resistant cultivarsor pathogen-free seed cane and quarantine [3 6] Con-ventional diagnostic approaches involve the application ofmorphological identification which is time consuming andthus not conducive to control the spread of disease forthe three months of disease period [3] or the isolationof the pathogen followed by biochemical identification andpathogenicity tests (requires more than one week) [7] orserological testing which requires high quality antibodies [8]or electron microscopy which needs expensive equipment[8] Until now none of sugarcane smut genome information

2 The Scientific World Journal

has been reported yet Albert and Schenck [9] had success-fully amplified smut with the bE4bE8 primers based onthe Ustilago maydis bE mating-type gene and its specificconventional PCR product (459 bp) (Genbank Accession noU612901) was found to be approximately 70 identical innucleotide sequence to the corresponding region of the bEgene inUstilagomaydis andUstilago hordei Southern analysisof S scitamineum revealed a specific hybridization with smutDNA rather than with uninfected sugarcane DNA or withDNA from Arthrobotrys musiformis PCR amplification fromthe bE gene was used as an assay for the presence of smutDNA and it was also validated by several other scientists[3 9] However one of the limitations of the conventionalPCRmethod is that they do not allow quantifying the amountof S scitamineum in the sugarcane and there is no report onsensitive detection and quantification assay for this pathogen

The exact detection and proper quantification of thesmut is therefore an essential requirement for an effectivemanagement practice Establishing a sensitive TaqMan real-time PCR detection method would be useful for quarantineepidemiological research and smut control This could beapplied to (i) sugarcane seeds or stalks importation andexport inspection for smut pathogen (ii) evaluation of smutresistance between or within several or a batch of sugarcanegenotypes by quantifying the copy number of smut pathogenin asymptomatic smut-infected sugarcane and (iii) super-vision and management of the pathogen-free seed cane ofsugarcane

In the present study a set of TaqMan real-time PCRprimers and a probe which were reliable and specific weredesigned according to the sequences of smut bE gene andused for the detection and quantification of smut in sugar-cane Here for the first time we report the development of aTaqMan real-time PCR assay for the smut which is comparedwith conventional PCR ismuchmore sensitive Based on thissystem the above three expected objectives were achievedFurthermore the teliospore germination process and thecurve of mycelial growth were observed in order to get abetter determination on the key timing of sugarcane-smutinteraction

2 Materials and Methods

21 Fungal Isolates and Growth Conditions Strain of sug-arcane-smut pathogen S scitamineum was selected for thedevelopment of TaqMan real-time PCR assay Smut-infectedwhips were collected from sugarcane cultivar ldquoROCrdquo22cultivated in the Key Laboratory of Sugarcane Biology andGenetic Breeding Ministry of Agriculture (Fuzhou China)Teliospores were mixed and sealed in plastic bags and thenstored at 4∘C This was used for gradient dilution in sterilewater and plated onto potato dextrose agar (PDA) containing75 120583gmL streptomycin (Shenggong China) Plates wereincubated in the dark for 4 d at 28∘C Single colonies weretransferred onto the new PDA medium and cultured at28∘C for 5 d Fungal mycelial DNA was extracted using theSDS method [10] and eluted with sterile water containing100 120583gmL RNase A The DNA sample was quantified using

the absorbance at 260 nm 280 nm by NanoVue plus (GEUSA) and stored at minus20∘C for later use

22 Primers and Probe Design and Specificity Test Thesequence of the smut bE gene was chosen as the PCRtarget gene for confirmation of the presence of smut asreported by Albert and Schenck [9] PCR amplificationwas performed in a total volume of 25120583L containing100 ng of smut DNA 400 nM each of the primersbE4 (51015840-CGCTCTGGTTCATCAACG-31015840) and bE8 (51015840-TGCTGTCGATGGAAGGTGT-31015840) 25 120583L 10 times 119864119909 TaqBuffer (Mg2+ Plus) 200120583M dNTPs and 0625U Ex TaqDNA polymerase (TaKaRa China) PCR comprised thefollowing steps 94∘C 4min 94∘C 55∘C and 72∘C 30 seach 35 cycles 72∘C 10min [9] The amplified bE fragment(459 bp) was purified from a 15 agarose gel and clonedinto Escherichia coli DH5120572 cell using pMD18-T vector(TaKaRa China) One positive clone was referred to aspbE The DNA of the recombinant plasmid was extractedusing Plasmid Mini Kit I (OMEGA China) and sent forsequencing (Shenggong China) in order to ensure that thetarget DNA sequence was amplified

The TaqMan probe (bEQ-P) and the primers (bEQ-FbEQ-R) were designed according to the sequence ofsmut bE gene using the Primer Express software ver-sion 20 (ABI Applied Biosystems USA) Primers bEQ-F (51015840-TGAAAGTTCTCATGCAAGCC-31015840) and bEQ-R (51015840-TGAGAGGTCGATTGAGGTTG-31015840) were designed to yielda 123 bp fragment of the bE gene The TaqMan probedbEQ-P (51015840-TGCTCGACGCCAATTCGGAG-31015840) contained6-carboxy-fluorescein (FAM) reporter dye at the 51015840 endand 6-carboxytetramethylrhodamine (TAMRA) fluorescentquencher at the 31015840 end The sequences used to designtesting primers and probe were compared to other organismsin National Center for Biotechnology Information (NCBIhttpwwwncbinlmnihgov) to confirm the specificity andwere commercially synthesized by TaKaRa Biotechnology(Dalian China)

The specificity of PCR-based TaqMan assay was per-formed using 100 ng DNA of S scitamineum Phoma spFusarium moniliforme Pestalotia ginkgo Fusarium oxys-porum and Helminthosporium sacchari Meanwhile caneexudates from sugarcane genotypes Yacheng05-179 (smutresistance) (private bulletin) and ldquoROCrdquo22 (smut susceptible)(private bulletin) were cultivated at PDA liquidmedium con-taining 75120583gmL streptomycin at 200 rpm at 28∘C overnightin order to obtain the endophytes respectively All eighttested fungal DNA preparations were extracted by SDSmethod

23 TaqMan Real-Time PCR Conditions and Standard CurvesTaqMan real-time PCR was performed in an ABI 7500 real-time PCR system (Applied Biosystems USA) Amplificationreaction contained 125 120583L of 2 times TaqMan Universal MasterMix (Roche Shanghai China) 10120583L each of 10 120583M primers(bEQ-F and bEQ-R) 02 120583L of 10120583M probe and 10 120583L oftemplate DNA and nuclease-free water to a final volumeof 25 120583L The thermal profile consisted of 50∘C for 2min

The Scientific World Journal 3

followed by 95∘C for 10min and 40 cycles of 95∘C for 15 s and60∘C for 1min Fluorescence was measured once per cycle atthe end of the 60∘C segment

The standard curve was generated using tenfold serialdilutions with ddH

2O from 10minus3 to 10minus10 (approximately

1987 times 108

sim 1987 times 101 copies120583L) of 100 ng120583L pbE DNA

The PCR conditions for this standard curve were adopted toperform the further reactions to estimate the copy number ofsmut pathogen by TaqMan real-time PCR assay

24 Sensitivity Comparisons of TaqMan Real-Time PCR andConventional PCR The sensitivity for the detection andquantification limits of the primers was investigated basedon the plasmid with a known copy number of the bE insertThe pbE DNA (100 ng120583L) was tenfold serial diluted 10minus3 to10minus10Thendilutionswere detected byTaqMan real-timePCRas described above The gene copy number was calculatedas follows CopiesmL = 602 times 1023 times (concentrationgmL)(MW gmol) MW = genome length times 660 daltonbp[11] In parallel the same samplewas detected by conventionalPCR method [9] Conventional PCR amplified product witha length of 459 bp was detected on 15 agarose gel The end-point dilution of these two assays in which a positive resultwas recorded was compared

In order to further evaluate the detection accuracy andsensitivity in sugarcane three pieces of the youngest fullyexpanded leaf namely +1 leaf which with a visible dewlap(the collar between the leaf blade and sheath) were collectedfrom smut-infected plants of 10 months old of cultivarldquoROCrdquo22 and used for detection analysis by both real-timePCR and conventional PCR assay Two microlitres of fivefoldserial dilutions (500 100 20 40 and 08 ng120583L) of +1 leafDNA was used as templates and TaqMan and conventionalPCR assay were performed in the protocols as describedabove

25 Germination Process and Growth Curve of SmutTeliospores As for understanding the biological char-acteristics of S sporisorium the germination process andthe growth curve of smut teliospores were observed Bygermination assay teliospores from smut whip were trans-ferred onto the PDA liquid medium containing 75 120583gmLstreptomycin and cultured at 140 rpm at 28∘CThe teliosporegermination periods were then monitored by microscopy(Axio ScopeA1 Germany) at 0 h 6 h 12 h 24 h 36 h and 48 hafter cultivation [12] For growth curve observation smutspores were eluted to 5 times 106 sporesmL with 300mL sterilePDA liquid medium (containing 75120583gmL streptomycin)and cultured at 140 rpm at 28∘C [10] During the cultureprocess a spectrophotometer (Lambda35 Perkin ElmerAmerica) was used to determine the optical density at600 nm at 25 time points at set intervals between 0 h and288 h The growth curves were drawn by OriginPro 80software with culture time as abscissa and OD

600value as

ordinate

26 Detection and Quantification of S sporisorium in Sugar-cane Plantlets Inoculatedwith Smut Sugarcane cultivar FN40

(a widely grown cultivar in China) was provided by theKey Laboratory of Sugarcane Biology and Genetic BreedingMinistry of Agriculture (Fuzhou China) The pathogen-free FN40 four-month-old plantlets were syringe-inoculatedfrom the basal portion up to 2 cm length with 05 120583L of thesmut suspension containing 5 times 106 sporesmL in 001 (vv)Tween-20 [7 13] All plantlets were incubated at 28∘C underconditions of 12 h light and 12 h darkness At each time pointof 0 h 12 h 24 h 48 h 120 h 168 h and 336 h after inoculationtreatment three culms were sampled for DNA extraction byCTAB-based protocol as reported [14] These DNA sampleswere subjected to the TaqMan assay

27 Smut Resistance Evaluation Using TaqMan Real-Time PCR Two-bud sets of the sugarcane genotypes ofYacheng05-179 (resistant) and ldquoROCrdquo22 (susceptible) wereinoculated with 05120583L smut suspension as described aboveAfter that five buds from both varieties were excised at 0 h12 h 24 h 48 h and 168 h after inoculation respectivelyCollected samples were washed with distilled water frozenin liquid nitrogen and stored at minus80∘C until extractionof DNA Quantification of smut pathogen in the budswas accomplished by calculating the target amplicon copynumber based on TaqMan real-time PCR

28 Statistical Analysis Each run of TaqMan real-time PCRcontained three replicates as well as three smut-DNA-template positive controls mock controls (pathogen-freeplantlets syringe-inoculated with sterile ddH

2O) and blank

controls (without template DNA) For the evaluation ofTaqMan assay efficiency and the calculation of the copy num-ber of the smut pathogen a standard curve was generatedusing pbE DNA in each TaqMan assay Data analyses wereperformed usingMicrosoft Excel andOriginPro 80 softwareStandard curve analysis was based on threshold cycles values(Ct) and serial dilutions of pbE DNA (10minus3 to 10minus10)The Δ119877

119899

(change in normalised fluorescence) records the amount ofthe product amplified Correlation coefficient (R2) slope (S)and efficiency (119864() = 10(1119878) minus 1) [15 16] of amplificationwere calculated to assess the linear range and reliability of theTaqMan real-time PCR assay By the equation of the linearregression line (119910 = 119886119909 + 119887) (a was the slope and b was theintercept) the copy number can be calculated in sugarcanesamples [17] The formula for the copy number was inferredfrom the equation copy number = 120582 times 10(Ctminus119887)119886 [18] In thisstudy 120582 was a fixed value as 1987 times 1011 for the dynamicrange of smut which was obtained by the copy number foreach serial dilution (100 pg rarr 10 ag) The Ct value below35 indicated a positive result [19] If the Ct value exceeds 35the sample should be detected again from which no Ct valuemeans negative otherwise positive

3 Results

31 Design of Primers and Probe ABLAST comparison of the123 bp region of the smut bE gene for which the primers weredesigned showed no similarity with other sequences but had


a 100 sequence identity with the target bE gene in GenBankFurthermore using BLAST the sequences of the primersand probe did not completely match any sequences of otherpublished organisms In all TaqMan real-time PCR reactionsno fluorescence was observed for the negative (mock andblank) samples but successful detection was observed inpositive controls (pbE and smut DNA) In addition speciesspecificity was evaluated by testing DNA of some commonencountered fungal diseases on sugarcane (Phoma sp Fusar-iummoniliforme Pestalotia ginkgo Fusarium oxysporum andHelminthosporium sacchari) and endophytes of Yacheng05-179 and ldquoROCrdquo22 by TaqMan assay under the same con-dition No positive result was observed in these samplesTherefore the primers bEQ-FbEQ-R and probe bEQ-P wereselected for further experimentation

32 Standard Curves and Amplification Efficiency In gen-erated standard curves linearity between the TaqMan real-time PCR Ct values and target concentration was observedover eight orders of magnitude in ten-fold serial dilutionsin triplicate (Figure 1) Initial quantities of smut pbE DNAtemplates were 100 pg 10 pg 1 pg 100 fg 10 fg 1 fg 100 agand 10 ag approximately 1987 times 108 to 1987 times 101 copiesrespectively Ct value ranged from 14 to 37 (Figure 1(a)) Alinear regression analysis (1198772 = 0998 119864() = 1032)which revealed a linear relationship between the quantities oftemplates andCt values was performedwithΔ119877

119899andCt and

the results were shown in Figure 1(b) which demonstratedthat the TaqMan real-time PCR protocol was feasible to smutpathogen quantification

33 Comparison of the Sensitivity of TaqMan Real-Time PCRAssay and Conventional PCR Eight ten-fold serial dilutionsof pbE DNA were assayed to determine the detection sensi-tivity by two different methods This assay showed excellentresults with regard to sensitivity compared to conventionalPCR Stable amplification was observed for as low as 10 agof pbE DNA in three replications of TaqMan real-timePCR assay (Figure 1) indicating the lowest limit of smutdetection by this assay was 1987times101 copies In conventionalPCR the expected amplicon of 459 bp was obtained in thereaction by detection on agarose gel and sequenced andthe detection limit was 10 fg which is equivalent to 1987 times104 copies (Figure 2) The above results demonstrated that

the application of TaqMan real-time PCR assay in relativelyaccurate quantification of the target DNAwas possible whichalso showed a wider dynamic range of nearly 1000 (1987 times104

1987 times 101) times more sensitivity than conventional

PCRTo further validate the sensitivity of this TaqManmethod

+1 leaf gDNA of ldquoROCrdquo22 infected with smut was seriallydiluted five fold andmeasured by TaqMan real-time PCR andconventional PCR The TaqMan assay R2 value was 0998 Ctvalues ranged from 28202plusmn0354 to 36575plusmn0330 (Table 1)The minimum detection limit was 08 ng120583L with mean Ctvalue of 36575 plusmn 0330 which was estimated to containaround 41123 plusmn 4953 copies of bE target gene (Table 1)When the concentration of the +1 leaf gDNA was reduced

Cycle403836343230282624222018161412108642

10

001

0001

01

000001

1

0000001

00001

0053687

ΔRn

(a)

000100001000001000000100000001000000001000000000100000000001

13

14

26

38

37

36

35

34

33

32

31

30

29

28

27

25

24

23

22

21

20

19

18

17

16

15

000000000001

Quantity

CT

(b)

Figure 1 Sensitivity test of TaqMan real-time PCR assay for ten foldserial dilutions of smut pbE DNA standard (a) The amplificationplots showed the testing results of a ten fold dilution serialscontaining the following pbE DNA 100 pg 10 pg 1 pg 100 fg 10 fg1 fg 100 ag and 10 ag while no amplification signals were observedin the mock and blank controls (b) The linear regression analysesbetween the quantities of templates and Ct values Regressionequations were calculated with 119910 = minus3247119909+4629 1198772 = 0998 119878 =minus3247 and 119864() = 1032

to 100 ng120583L no apparent amplification was observed onagarose gel (Figure 3)These results revealed that the TaqManassay developed in this study was more sensitive than that ofconventional PCR

34 Germination Process and Growth Curve of SmutTeliospores The observation of teliospore germinationprocess by microscopy is shown in Figure 4(a) Sporesbegan to germinate after 6 h after inoculation and sproutedconsiderably at 12 h The process of spore germination isone germ tube producing one promycelium The promyceliawere found and began to detach at 12 h There were manyfree promycelia at 24 h and these promycelia lengthened


Table 1 Mean Ct values and copy numbers obtained by sensitivity test by TaqMan real-time PCR assay

Concentration of +1 leaf DNA (ng120583L) Ct values (Ma plusmn 120590) Copy numbers (Ma plusmn 120590) (copies120583L)500 28202 plusmn 0354 12247062 plusmn 1579742

100 30700 plusmn 0391 2246733 plusmn 349973

20 32895 plusmn 0456 509013 plusmn 101842

4 35561 plusmn 0200 81431 plusmn 2540

08 36575 plusmn 0330 41123 plusmn 4953

Ma = mean value of three technical replicates 120590 = standard error

M 888777666555444333222111

Figure 2 Sensitivity test of conventional PCR based on eight ten-fold serial dilutions of smut pbE DNA M 100 bp DNA laddermarker Lane 1 100 pg120583L Lane 2 10 pg120583L Lane 3 1 pg120583L Lane 4100 fg120583L Lane 5 10 fg120583L Lane 6 1 fg120583L Lane 7 100 ag120583L Lane8 10 ag120583L

M 888777666555444333222111

Figure 3 Sensitivity test of conventional PCR based on five-foldserial dilutions of +1 leaf gDNAM 100 bpDNA laddermarker Lane1 500 ng120583L Lane 2 100 ng120583L Lane 3 20 ng120583L Lane 4 4 ng120583LLane 5 08 ng120583L Lane 6 positive control Lane 7 mock controlLane 8 blank control

as the culturing continued Some promycelia outgrewbasidiospores at 36 h and a small amount of them beganto detach to form microspores At 48 h a large number ofspores had detached which lead to many microspores andresulted in turbidity of the liquid medium In parallel regularsamples were measured at OD

600 nm (Figure 4(b)) A slowphase of smut pathogen growth was observed from 0 h to12 h then a logarithmic growth phase appeared from 12 hto 60 h followed by a stationary phase and declining phaseBased on the above results the germination process wassummarized as follows teliospore germination and thengerm tube promycelium basidiospores and microsporesproduced one by oneThe growth curve could be divided intoslow phase (0ndash12 h) logarithmic growth phase (12ndash60 h)

and stationary phase (60 h-) This will help to understandthe correlation between germination and mycelial growthand to further determine the key interaction time of thesugarcane-smut relationshipTherefore during the followingartificially inoculation treatments 12 h 24 h 48 h 120 h168 h and 336 h were determined to be the sampling timepoints

35 Testing of Smut-Infected Sugarcane Plantlets Using the Taq-Man Real-Time PCR Assay In order to detect the dynamicrange of the assay on infected sugarcane pathogen-freeplantlets of variety FN40 were artificially inoculated with thesmut pathogen and genomic DNA extracted from samples at12 h 24 h 48 h 120 h 168 h and 336 h The sample collectedat 0 h was used as the mock control The standard curve wasgenerated using pbE DNA with the R2 value in the linearregression being 0994 Among these inoculated samples Ctvalues ranged from 28084 plusmn 0022 to 35214 plusmn 0034 and thequantity of smut varied from 77695 plusmn 6177 to 12148273 plusmn473911 (Figure 5) Smut pathogen could be detected withinthe first 12 h At 12 h 77695 plusmn 6177 copies120583L of smutpathogen was detected and the corresponding fluorescentlevels continued to increase from 24 h (456946 plusmn 50077copies120583L) to 168 h (12148273plusmn473911 copies120583L) howeverthey dropped remarkably at 336 h (3140044 plusmn 315611copies120583L) It meant that compared with the copy numberof S scitamineum at 12 h in FN40 dynamic 59- 103- 1268-and 1564-fold increasingwas detected at 24 h 48 h 120 h and168 h respectively The copy numbers decreased by 742 at336 h compared to those at 168 h but still higher than thoseof 12 h

36 Copy Numbers Assay in Sugarcane Resistant and Suscep-tible Genotypes Challenged with S scitamineum Sugarcanegenotypes Yacheng05-179 (smut resistant) and ldquoROCrdquo22(smut susceptible) were artificially inoculated in the budsand used for investigation of the correlation between copynumber of S scitamineum and smut resistance The R2 ofthe standard curve was 0998 for TaqMan PCR Figure 6showed that the results of the smut pathogen quantificationand the difference of copy numbers between resistant andsusceptible varieties were significant It should be notedthat no amplification was observed in the mock and blankcontrols After challenge the copy numbers for resistantvariety ranged from 43761 plusmn 5464 to 349772 plusmn 72078 withthe lowest at 12 h (43761 plusmn 5464) and the highest at 24 h(349772 plusmn 72078) and dropped to 55569 plusmn 721604 at 168 h


0h 6h 12h

24h 36h 48h

(a)

00

05

10

15

20

25

30

35

OD

val

ue

2882642402161921801681561441321201089684724836302412963Culture time (h)

0 60

(b)

Figure 4 Germination (a) and growth curve (b) of smut teliospores (a) Teliospore germination monitored by microscopy (magnificationtimes400) Bar = 20 120583m (b) The growth curve during the culture process (from 0 h to 288 h) all data points mean plusmn SE (119899 = 3)

In contrast copies in susceptible variety varied from205658 plusmn 94320 to 20556141 plusmn 1384162 with much higherconidial densities than those of the resistant variety especiallyat first sampling point (545402plusmn80111) and at last samplingpoint (20556141 plusmn 1384162) indicating that susceptiblevariety was more easily infected by the pathogen and moreconducive to the pathogen proliferation than the resistantvariety

4 Discussion

The detection of smut pathogens is important at the earlystages of sugarcane colonization [20] since it is difficult to dif-ferentiate it from other fungi based on mycelial morphologyThe production of smut sori was observed during 6ndash12 weeksafter pathogen challenge PCR combined with microscopicexamination was used to investigate smut infection in sugar-cane during this period with fungal hyphae beginning to befound 8 weeks after inoculation by microscopy [7] Howeverearly detection of target pathogen is essential for assessing the

health status of plants before the transplanting of plantletsor seedlings into the field [21] Schenck [22] found thatconventional PCR assays were significantly more sensitiveand efficient than microscopy for smut pathogen detectionSimilarly Singh et al [7] found that the smut pathogen couldbe detected at 12 h after challenge by PCR with primersbE4bE8 indicating that the PCR assay was more sensitivethan that of microscopy Recently the specificity of PCRamplification to bE gene target of S scitamineum has beenvalidated successfully by several other researchers [3 7]Although visible in detecting the presence of the pathogenconventional PCR was insufficient to quantify the pathogenin nature Due to its high specificity sensitivity accuracyand speed real-time PCR is a suitable detection technique[23] This technique including TaqMan real-time PCR hasbeen widely used for the diagnosis of pathogens such asplanta botrytis cinerea [24] sugarcane yellow leaf virus [19]maize chlorotic mottle virus [8] and cucumber vein yellowingvirus [25] Here we describe the first report of TaqMan real-time protocol for the sensitive detection and quantification of


0

1000

2000

3000

10000

12000

14000

3361681204824Inoculation time (h)

FN40

12

Cop

y nu

mbe

rs o

f sm

ut (c

opie

s120583

L)

Figure 5Detection of smut-infected FN40plantlets Copynumbersof smut were calculated with the equation of the linear regressionline All data points mean plusmn SE (119899 = 3)

0

1000

2000

3000

4000

20000

22500

25000

ResistantSusceptible

Inoculation time (h)168482412

Cop

y nu

mbe

rs o

f sm

ut (c

opie

s120583

L)

Figure 6 Quantification of the pathogen in sugarcane genotypeschallenged with S scitamineum Resistant Yacheng05-179 suscep-tible ldquoROCrdquo22 Copy numbers of smut were calculated with theequation of the linear regression line All data points mean plusmn SE(119899 = 3)

S scitamineum in sugarcane The specificity of the TaqManprobe and primers designed according to the sequences ofsmut bE gene was confirmed by TaqMan real-time PCR andthe corresponding PCR product was sequenced and com-pared to the databases using the BLAST tool Meanwhile thespecificity of our probe was again determined by searchingthe nucleotide databasesThere was no significantmatchwithany sequence from other organisms Furthermore successful

detection of smut was achieved in pbE DNA smut DNA andsugarcane samples As there was not any report of closelyrelated genus with smut the common encountered fungaldisease on sugarcane was chosen for detection Nonpositiveresult of species-specificity test which was evaluated byDNA samples of Phoma sp Fusariummoniliforme Pestalotiaginkgo Fusarium oxysporum andHelminthosporium sacchariand endophytes of Yacheng05-179 and ldquoROCrdquo22 suggestedthe good specificity of designed primers and probe for thesmut strain and sugarcane

With the development of the PCR technique the requireddetection sensitivity could be achieved This method ismore convenient than serological technique and hybridiza-tion assays using DNA probes which are time consuminginsensitive and additional technical skills are required Ithas been reported that conventional PCR is not suitablefor detection of numerous samples by running gels andmay even be polluted during post-PCR operation [26] withsensitivity much lower than that of real-time PCR The mainadvantages of TaqMan real-time PCR assay are its highsensitivity and reliability (one step and gel free) As reportedbefore TaqMan real-time PCR method could detect up to4 fg DNA of Mycobacterium avium subsp paratuberculosis[27] The results obtained in the present study revealed thatboth the primer pair of bEQ-FbEQ-R and the TaqMan probewere specific and sensitive to smut pathogen (Figures 1 2 and3) and the detection limit for the TaqMan assay was 10 ag(1987times101 copies) of pbEDNA and 10 fg (1987times104 copies)for the conventional PCR indicating that the sensitivity ofthe TaqMan assay was 1000 times than that of conventionalPCR (Figures 1 and 2) Further to evaluate the specificityand sensitivity of the TaqMan assay developed in this studywe also applied it to detect the smut in sugarcane DNA andgained a result with 08 ng (41123 plusmn 4953 copies120583L) of +1leaf gDNA of ldquoROCrdquo22 infected with smut pathogen whichwas 125 times more sensitive than that of conventional PCR(Table 1 and Figure 3)

Smut spores germinated on the sugarcane internodalsurface and it was followed by the formation of appressoriaon the inner scales of the young buds and on the base ofthe emerging leaves Entry into the bud meristem occurredbetween 6 and 36 h after the teliospore deposition [5] In thepresent study we carried out the experiments of spore germi-nation and growth curve observation (Figure 4) in order toestablish the correlation between the time of germination andthe time of smut mycelial growth which should lead to a bet-ter determination on the key interaction time of sugarcane-smut biosystem The results indicated that the germinationprocess of S scitamineum was as follows teliospores germi-nate germ tube promycelium basidiospore andmicrosporewhich was similar to the observation of mycelial growthprocess of Sporisorium reilianum in maize seedling by Zhanget al [28] The growth curve of smut pathogen could bedivided into slow growth phase (0ndash12 h) logarithmic growthphase (12ndash60 h) and stationary growth phase (60 h-) Basedon the above results the time points of 12 h 24 h 48 120 h168 h and 336 h after smut pathogen challenge were chosenas sampling times


Asmore andmore sequence datawas available for design-ing primers and probes for specific detection of pathogensthere is no doubt that real-time PCR will become a routinetechnique in the plant quarantine area [29] Sugarcane smutis one of the most prevalent diseases affecting sugarcaneyield and can cause considerable economic losses [30ndash32] As reported correct quarantine smut resistant varietyreleasing and integrated field management are the threemain strategies to control smut disease [3] Due to a hugeamount of spores released by smut whips it is difficultto stop the infection or reinfection of sugarcane includingpathogen-free seedlings or plantlets Pathogen detection isa crucial procedure in the import and export of sugarcanestalk during germplasm exchange and in the supervision andmanagement of pathogen-free cane or plantlets from tissuecultureHowever there is not any report about smut pathogendetection in sugarcane pathogen-free seedlings In this studythe TaqMan assay was successfully applied to quantify thesmut pathogen in tissue cultured plants (FN40) challengedby the pathogen (Figure 5) and the results indicated thatthis assay was capable to detect the pathogen at the earlystage (12 h) of the challenge and at the limit of 456946 plusmn50077 copies The new TaqMan real-time PCR technologysystem can be used to assess whether the sugarcane seed caneseedlings or plantlets are really smut pathogen-free Thus itis useful in the production and supervision of pathogen-freesugarcane seed cane in the programme of pathogen-free seedcane in mainland China

Smut resistance is an important agronomic trait due tothe serious loss in sugarcane stalk yield caused by smutpathogen [6 31 32] Based on the TaqMan PCR assay thecopy numbers of the pathogen at 12ndash168 h in smut resistantvariety Yacheng05-179 challenged by S scitamineum weremuch lower (ranging from 43761 plusmn 5464 to 349772 plusmn72078) than those of susceptible variety ldquoROCrdquo22 (rangingfrom 205658 plusmn 94320 to 20556141 plusmn 1384162) (Figure 6)suggesting the TaqMan PCR assay system developed in thisstudy might be used for smut resistance evaluation if morevalidation was performed This TaqMan PCR assay systemcan be used for evaluation of smut resistance in two severalor a batch of sugarcane genotypes based on the resultsachieved The current method of smut resistance evaluationis still both time and field consuming using observationof smut whip in at least two sugarcane crops [3 33ndash35]In addition the sensitive and accurate quantification of thesmut pathogen by TaqMan PCR assay is beneficial in givinginsight into the mechanisms of sugarcane-smut pathogeninteraction

In summary the present study confirmed that thesedesigned primer sets and probe are highly specific andsensitive for smut detection The TaqMan real-time PCRassay established here can shorten testing time and be usedas a tool for the detection and quantification of this pathogenin sugarcane Its advantages are those as follows (i) ensur-ing pathogen-free sugarcane seeds or stalks imported andexported through the sensitive detection of smut pathogen(ii) providing a new insight into the evaluation of smutresistance of sugarcane genotypes by quantifying the copynumbers of smut pathogen in asymptomatic smut-infected

sugarcane and (iii) gaining supervision and efficientmanage-ment of pathogen-free sugarcane

Acknowledgments

This work was funded by National Natural Science Founda-tion of China (no 31101196) the earmarked fund for theMod-ern Agriculture Technology of China (CARS-20) ResearchFunds for Distinguished Young Scientists in Fujian Agricul-ture and Forestry University (xjq201202) and National HighTechnology Research and Development Program of China(863 Program) Project (2013AA102604) The authors espe-cially thank Andrew C Allan in The New Zealand Institutefor Plant amp Food Research Ltd (Plant and Food Research)Mt Albert Research Centre Auckland New Zealand for hiscritical revision and valuable comments on this paper

References

[1] P Padmanaban K C Alexander and N Shanmugan ldquoEffectof smut on growth and yield parameters of sugarcanerdquo IndianPhytopathology vol 41 no 4 pp 367ndash369 1988

[2] Y X Que L P Xu J W Lin R K Chen and M P GrishamldquoMolecular variation of Sporisorium scitamineum in MainlandChina revealed byRAPDandSRAPmarkersrdquoPlantDisease vol96 no 10 pp 1519ndash1525 2012

[3] A R Sundar E L Barnabas PMalathi and R Viswanathan ldquoAmini-review on smut disease of sugarcane caused by Sporiso-rium scitamineumrdquo in Botany J Mworia Ed pp 109ndash128InTech Rijeka Croatia 2012

[4] V P Agnihotri Diseases of Sugarcane and Sugarbeet Oxford ampIBH New Delhi India 1990

[5] K C Alexander and K Ramakrishnan ldquoInfection of the budestablishment in the host and production of whips in sugarcanesmut (Ustilago scitaminea) of sugarcanerdquo Proceedings of theInternational Society For Sugarcane Technology vol 17 pp 1452ndash1455 1980

[6] L P Xu RKChen andPHChen ldquoAnalysis on infection indexof smut caused by Ustilago scitaminea in sugarcane segregatedpopulationrdquo Chinese Journal of Tropical Crops vol 25 no 3 pp33ndash36 2004

[7] N Singh B M Somai and D Pillay ldquoSmut disease assessmentby PCR andmicroscopy in inoculated tissue cultured sugarcanecultivarsrdquo Plant Science vol 167 no 5 pp 987ndash994 2004

[8] Y Zhang W Zhao M Li H Chen S Zhu and Z Fan ldquoReal-time TaqMan RT-PCR for detection of maize chlorotic mottlevirus in maize seedsrdquo Journal of Virological Methods vol 171no 1 pp 292ndash294 2011

[9] H H Albert and S Schenck ldquoPCR amplification from ahomolog of the bE mating-type gene as a sensitive assay for thepresence ofUstilago scitamineaDNArdquo Plant Disease vol 80 no10 pp 1189ndash1192 1996

[10] Y X Que T T Zou and L P Xu ldquoThe screening of mediumfor sugarcane Ustilago scitaminea and the technique for theisolation of its genomic DNArdquo Acta Agriculturae UniversitatisJiangxiensis vol 26 no 3 pp 353ndash355 2004

[11] U J Ciglenecki and I Toplak ldquoDevelopment of a real-time RT-PCR assay with TaqMan probe for specific detection of acutebee paralysis virusrdquo Journal of Virological Methods vol 184 no1-2 pp 63ndash68 2012


[12] J Pairoj and W Suwanit ldquoEffect of temperature and somesubstrate on teliospore germination of sugarcane smut andinoculation methods on infection by Ustilago scitamineardquoKasetsart Journal vol 17 pp 45ndash52 1983

[13] L N Thokoane and R S Rutherford ldquocDNA-AFLP differentialdisplay of sugarcane (Saccharum spp hybrids) genes inducedby challenge with the fungal pathogen Ustilago scitaminea(sugarcane smut)rdquo Proceedings of the South African SugarTechnologists Association vol 75 pp 104ndash107 2011

[14] W Yao A L Yu J S Xu H ZhouMQ Zhang and R K ChenldquoA simple and quick method for extracting sugarcane genomicDNArdquo Journal of Agricultural Biotechnology vol 12 no 1 pp121ndash122 2005

[15] M W Pfaffl ldquoQuantification strategies in real-time PCRrdquo in A-Z of Quantitative PCR S A Bustin Ed pp 87ndash113 IUL 2004

[16] R G Rutledge and C Cote ldquoMathematics of quantitativekinetic PCR and the application of standard curvesrdquo NucleicAcids Research vol 31 no 16 pp 93ndash93 2003

[17] S A Bustin ldquoAbsolute quantification of mrna using real-timereverse transcription polymerase chain reaction assaysrdquo Journalof Molecular Endocrinology vol 25 no 2 pp 169ndash193 2000

[18] M Pilotti V Lumia G Di-Lernia and A Brunetti ldquoDevelop-ment of Real-Time PCR for in wood-detection of Ceratocystisplatani the agent of canker stain of Platanus spprdquo EuropeanJournal of Plant Pathology vol 134 no 1 pp 61ndash79 2012

[19] J Korimbocus D Coates I Barker and N BoonhamldquoImproved detection of Sugarcane yellow leaf virus using a real-time fluorescent (TaqMan)RT-PCR assayrdquo Journal of VirologicalMethods vol 103 no 2 pp 109ndash120 2002

[20] H L Lloyd and G Naidoo ldquoA qualitative semi-automatedtechnique for the assessment of smut colonization of sugarcanestalk tissue prior to whip formationrdquo Sugarcane PathologistsrsquoNewsletter vol 26 pp 48ndash51 1981

[21] M H Kim M S Cho B K Kim et al ldquoQuantitative real-time polymerase chain reaction assay for detection of Pectobac-terium wasabiae using YD repeat protein gene-based primersrdquoPlant Disease vol 96 no 2 pp 253ndash257 2012

[22] S Schenck ldquoEvaluation of a PCR amplification method fordetection of systemic smut infections in sugarcanerdquo Sugar Canevol 6 pp 2ndash5 1998

[23] K A La Fauce R Layton and L Owens ldquoTaqMan real-time PCR for detection of hepatopancreatic parvovirus fromAustraliardquo Journal of Virological Methods vol 140 no 1-2 pp10ndash16 2007

[24] M B Suarez KWalsh N Boonham T OrsquoNeill S Pearson andI Barker ldquoDevelopment of real-time PCR (TaqMan) assays forthe detection and quantification of Botrytis cinerea in plantardquoPlant Physiology and Biochemistry vol 43 no 9 pp 890ndash8992005

[25] F M Gil-Salas A Colyer N Boonham I M Cuadradoand D Janssen ldquoResistance screening against Cucumber veinyellowing virus using a real-time (Taqman) RT-PCR assay incucumber (Cucumis sativus)rdquoCrop Protection vol 28 no 1 pp109ndash112 2009

[26] N W Schaad and R D Frederick ldquoReal-time PCR andits application for rapid plant disease diagnosticsrdquo CanadianJournal of Plant Pathology vol 24 no 3 pp 250ndash258 2002

[27] S V Ravva and L H Stanker ldquoReal-time quantitative PCRdetection ofMycobacterium avium subsp paratuberculosis anddifferentiation from other mycobacteria using SYBRGreen andTaqMan assaysrdquo Journal of Microbiological Methods vol 63 no3 pp 305ndash317 2005

[28] B Y Zhang Z G Gao J H Zhuang X F Zhang and H ZhaoldquoThe mycelium growth process of Sporisorium Relianum inmaize seedlingrdquo Seed vol 27 no 6 pp 8ndash9 2008

[29] CHongyun ZWenjun G Qinsheng C Qing L Shiming andZ Shuifang ldquoReal time TaqManRT-PCR assay for the detectionof Cucumber green mottle mosaic virusrdquo Journal of VirologicalMethods vol 149 no 2 pp 326ndash329 2008

[30] J W Hoy C A Hollier D B Fontenot and L B GrelenldquoIncidence of sugarcane smut in Louisiana and its effects onyieldrdquo Plant Disease vol 70 no 1 pp 59ndash60 1986

[31] Y X Que J W Lin X X Song L P Xu and R K ChenldquoDifferential gene expression in sugarcane in response tochallenge by fungal pathogen Ustilago scitaminea revealed bycDNA-AFLPrdquo Journal of Biomedicine and Biotechnology vol2011 Article ID 160934 10 pages 2011

[32] Y Que L Xu J Lin M Ruan M Zhang and R Chen ldquoDif-ferential protein expression in sugarcane during sugarcane-sporisorium scitamineum interaction revealed by 2-DE andMALDI-TOF-TOFMSrdquo Comparative and Functional Ge-nomics vol 2011 Article ID 989016 10 pages 2011

[33] D M Gong and R K Chen ldquoAdvances in the study on resis-tance mechanism and heritance to sugarcane smut caused byUstilago scitamineardquo Journal of Fujian Academy of AgriculturalSciences vol 10 no 1 pp 33ndash36 1995

[34] Y X Que L P Xu J W Lin T S Chen R K Chen and Y LLi ldquoEstablishment of evaluation system of smut resistance forsugarcane varietiesrdquo Journal of Plant Genetic Resources vol 7no 1 pp 18ndash23 2006

[35] L P Xu Y Q Lin and H Y Fu ldquoEvaluation of smut resistancein sugarcane and identification of resistance in sugarcanevarietiesrdquo Journal of Fujian Agricultural University vol 29 no3 pp 292ndash295 2000

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

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


has been reported yet Albert and Schenck [9] had success-fully amplified smut with the bE4bE8 primers based onthe Ustilago maydis bE mating-type gene and its specificconventional PCR product (459 bp) (Genbank Accession noU612901) was found to be approximately 70 identical innucleotide sequence to the corresponding region of the bEgene inUstilagomaydis andUstilago hordei Southern analysisof S scitamineum revealed a specific hybridization with smutDNA rather than with uninfected sugarcane DNA or withDNA from Arthrobotrys musiformis PCR amplification fromthe bE gene was used as an assay for the presence of smutDNA and it was also validated by several other scientists[3 9] However one of the limitations of the conventionalPCRmethod is that they do not allow quantifying the amountof S scitamineum in the sugarcane and there is no report onsensitive detection and quantification assay for this pathogen

The exact detection and proper quantification of thesmut is therefore an essential requirement for an effectivemanagement practice Establishing a sensitive TaqMan real-time PCR detection method would be useful for quarantineepidemiological research and smut control This could beapplied to (i) sugarcane seeds or stalks importation andexport inspection for smut pathogen (ii) evaluation of smutresistance between or within several or a batch of sugarcanegenotypes by quantifying the copy number of smut pathogenin asymptomatic smut-infected sugarcane and (iii) super-vision and management of the pathogen-free seed cane ofsugarcane

In the present study a set of TaqMan real-time PCRprimers and a probe which were reliable and specific weredesigned according to the sequences of smut bE gene andused for the detection and quantification of smut in sugar-cane Here for the first time we report the development of aTaqMan real-time PCR assay for the smut which is comparedwith conventional PCR ismuchmore sensitive Based on thissystem the above three expected objectives were achievedFurthermore the teliospore germination process and thecurve of mycelial growth were observed in order to get abetter determination on the key timing of sugarcane-smutinteraction

2 Materials and Methods

21 Fungal Isolates and Growth Conditions Strain of sug-arcane-smut pathogen S scitamineum was selected for thedevelopment of TaqMan real-time PCR assay Smut-infectedwhips were collected from sugarcane cultivar ldquoROCrdquo22cultivated in the Key Laboratory of Sugarcane Biology andGenetic Breeding Ministry of Agriculture (Fuzhou China)Teliospores were mixed and sealed in plastic bags and thenstored at 4∘C This was used for gradient dilution in sterilewater and plated onto potato dextrose agar (PDA) containing75 120583gmL streptomycin (Shenggong China) Plates wereincubated in the dark for 4 d at 28∘C Single colonies weretransferred onto the new PDA medium and cultured at28∘C for 5 d Fungal mycelial DNA was extracted using theSDS method [10] and eluted with sterile water containing100 120583gmL RNase A The DNA sample was quantified using

the absorbance at 260 nm 280 nm by NanoVue plus (GEUSA) and stored at minus20∘C for later use

22 Primers and Probe Design and Specificity Test Thesequence of the smut bE gene was chosen as the PCRtarget gene for confirmation of the presence of smut asreported by Albert and Schenck [9] PCR amplificationwas performed in a total volume of 25120583L containing100 ng of smut DNA 400 nM each of the primersbE4 (51015840-CGCTCTGGTTCATCAACG-31015840) and bE8 (51015840-TGCTGTCGATGGAAGGTGT-31015840) 25 120583L 10 times 119864119909 TaqBuffer (Mg2+ Plus) 200120583M dNTPs and 0625U Ex TaqDNA polymerase (TaKaRa China) PCR comprised thefollowing steps 94∘C 4min 94∘C 55∘C and 72∘C 30 seach 35 cycles 72∘C 10min [9] The amplified bE fragment(459 bp) was purified from a 15 agarose gel and clonedinto Escherichia coli DH5120572 cell using pMD18-T vector(TaKaRa China) One positive clone was referred to aspbE The DNA of the recombinant plasmid was extractedusing Plasmid Mini Kit I (OMEGA China) and sent forsequencing (Shenggong China) in order to ensure that thetarget DNA sequence was amplified

The TaqMan probe (bEQ-P) and the primers (bEQ-FbEQ-R) were designed according to the sequence ofsmut bE gene using the Primer Express software ver-sion 20 (ABI Applied Biosystems USA) Primers bEQ-F (51015840-TGAAAGTTCTCATGCAAGCC-31015840) and bEQ-R (51015840-TGAGAGGTCGATTGAGGTTG-31015840) were designed to yielda 123 bp fragment of the bE gene The TaqMan probedbEQ-P (51015840-TGCTCGACGCCAATTCGGAG-31015840) contained6-carboxy-fluorescein (FAM) reporter dye at the 51015840 endand 6-carboxytetramethylrhodamine (TAMRA) fluorescentquencher at the 31015840 end The sequences used to designtesting primers and probe were compared to other organismsin National Center for Biotechnology Information (NCBIhttpwwwncbinlmnihgov) to confirm the specificity andwere commercially synthesized by TaKaRa Biotechnology(Dalian China)

The specificity of PCR-based TaqMan assay was per-formed using 100 ng DNA of S scitamineum Phoma spFusarium moniliforme Pestalotia ginkgo Fusarium oxys-porum and Helminthosporium sacchari Meanwhile caneexudates from sugarcane genotypes Yacheng05-179 (smutresistance) (private bulletin) and ldquoROCrdquo22 (smut susceptible)(private bulletin) were cultivated at PDA liquidmedium con-taining 75120583gmL streptomycin at 200 rpm at 28∘C overnightin order to obtain the endophytes respectively All eighttested fungal DNA preparations were extracted by SDSmethod

23 TaqMan Real-Time PCR Conditions and Standard CurvesTaqMan real-time PCR was performed in an ABI 7500 real-time PCR system (Applied Biosystems USA) Amplificationreaction contained 125 120583L of 2 times TaqMan Universal MasterMix (Roche Shanghai China) 10120583L each of 10 120583M primers(bEQ-F and bEQ-R) 02 120583L of 10120583M probe and 10 120583L oftemplate DNA and nuclease-free water to a final volumeof 25 120583L The thermal profile consisted of 50∘C for 2min





1987 times 108






600value as

ordinate





2O) and blank


119899


3 Results





119899andCt and







Cycle403836343230282624222018161412108642

10

001

0001

01

000001

1

0000001

00001

0053687

ΔRn

(a)

000100001000001000000100000001000000001000000000100000000001

13

14

26

38

37

36

35

34

33

32

31

30

29

28

27

25

24

23

22

21

20

19

18

17

16

15

000000000001

Quantity

CT

(b)







100 30700 plusmn 0391 2246733 plusmn 349973

20 32895 plusmn 0456 509013 plusmn 101842

4 35561 plusmn 0200 81431 plusmn 2540

08 36575 plusmn 0330 41123 plusmn 4953


M 888777666555444333222111


M 888777666555444333222111








0h 6h 12h

24h 36h 48h

(a)

00

05

10

15

20

25

30

35

OD

val

ue

2882642402161921801681561441321201089684724836302412963Culture time (h)

0 60

(b)



4 Discussion




0

1000

2000

3000

10000

12000

14000


FN40

12

Cop

y nu

mbe

rs o

f sm

ut (c

opie

s120583

L)


0

1000

2000

3000

4000

20000

22500

25000



Cop

y nu

mbe

rs o

f sm

ut (c

opie

s120583

L)











Acknowledgments


References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





1987 times 108






600value as

ordinate





2O) and blank


119899


3 Results





119899andCt and







Cycle403836343230282624222018161412108642

10

001

0001

01

000001

1

0000001

00001

0053687

ΔRn

(a)

000100001000001000000100000001000000001000000000100000000001

13

14

26

38

37

36

35

34

33

32

31

30

29

28

27

25

24

23

22

21

20

19

18

17

16

15

000000000001

Quantity

CT

(b)







100 30700 plusmn 0391 2246733 plusmn 349973

20 32895 plusmn 0456 509013 plusmn 101842

4 35561 plusmn 0200 81431 plusmn 2540

08 36575 plusmn 0330 41123 plusmn 4953


M 888777666555444333222111


M 888777666555444333222111








0h 6h 12h

24h 36h 48h

(a)

00

05

10

15

20

25

30

35

OD

val

ue

2882642402161921801681561441321201089684724836302412963Culture time (h)

0 60

(b)



4 Discussion




0

1000

2000

3000

10000

12000

14000


FN40

12

Cop

y nu

mbe

rs o

f sm

ut (c

opie

s120583

L)


0

1000

2000

3000

4000

20000

22500

25000



Cop

y nu

mbe

rs o

f sm

ut (c

opie

s120583

L)











Acknowledgments


References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




119899andCt and







Cycle403836343230282624222018161412108642

10

001

0001

01

000001

1

0000001

00001

0053687

ΔRn

(a)

000100001000001000000100000001000000001000000000100000000001

13

14

26

38

37

36

35

34

33

32

31

30

29

28

27

25

24

23

22

21

20

19

18

17

16

15

000000000001

Quantity

CT

(b)







100 30700 plusmn 0391 2246733 plusmn 349973

20 32895 plusmn 0456 509013 plusmn 101842

4 35561 plusmn 0200 81431 plusmn 2540

08 36575 plusmn 0330 41123 plusmn 4953


M 888777666555444333222111


M 888777666555444333222111








0h 6h 12h

24h 36h 48h

(a)

00

05

10

15

20

25

30

35

OD

val

ue

2882642402161921801681561441321201089684724836302412963Culture time (h)

0 60

(b)



4 Discussion




0

1000

2000

3000

10000

12000

14000


FN40

12

Cop

y nu

mbe

rs o

f sm

ut (c

opie

s120583

L)


0

1000

2000

3000

4000

20000

22500

25000



Cop

y nu

mbe

rs o

f sm

ut (c

opie

s120583

L)











Acknowledgments


References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




100 30700 plusmn 0391 2246733 plusmn 349973

20 32895 plusmn 0456 509013 plusmn 101842

4 35561 plusmn 0200 81431 plusmn 2540

08 36575 plusmn 0330 41123 plusmn 4953


M 888777666555444333222111


M 888777666555444333222111








0h 6h 12h

24h 36h 48h

(a)

00

05

10

15

20

25

30

35

OD

val

ue

2882642402161921801681561441321201089684724836302412963Culture time (h)

0 60

(b)



4 Discussion




0

1000

2000

3000

10000

12000

14000


FN40

12

Cop

y nu

mbe

rs o

f sm

ut (c

opie

s120583

L)


0

1000

2000

3000

4000

20000

22500

25000



Cop

y nu

mbe

rs o

f sm

ut (c

opie

s120583

L)











Acknowledgments


References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0h 6h 12h

24h 36h 48h

(a)

00

05

10

15

20

25

30

35

OD

val

ue

2882642402161921801681561441321201089684724836302412963Culture time (h)

0 60

(b)



4 Discussion




0

1000

2000

3000

10000

12000

14000


FN40

12

Cop

y nu

mbe

rs o

f sm

ut (c

opie

s120583

L)


0

1000

2000

3000

4000

20000

22500

25000



Cop

y nu

mbe

rs o

f sm

ut (c

opie

s120583

L)











Acknowledgments


References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


0

1000

2000

3000

10000

12000

14000


FN40

12

Cop

y nu

mbe

rs o

f sm

ut (c

opie

s120583

L)


0

1000

2000

3000

4000

20000

22500

25000



Cop

y nu

mbe

rs o

f sm

ut (c

opie

s120583

L)











Acknowledgments


References












































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology






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 a taqman real-time pcr assay for...

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