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Journal of Virological Methods 183 (2012) 57– 62

Contents lists available at SciVerse ScienceDirect

Journal of Virological Methods

jou rn al h om epage: www.elsev ier .com/ locate / jv i romet

multiplex reverse transcription PCR assay for simultaneous detection of fiveobacco viruses in tobacco plants

in Daia, Julong Chengb, Ting Huanga, Xuan Zhengc, Yunfeng Wua,∗

State Key Laboratory of Crop Stress Biology in Arid Areas and Key Laboratory of Crop Pest Integrated Pest Management on the Loess Plateau of Ministry of Agriculture, College oflant Protection, Northwest A&F University, Yangling, 712100, ChinaShaanxi Province Tobacco Institute, Xi’an 710061, ChinaCollege of Life Science and State Key Laboratory of Crop Stress Biology in Arid Areas, Northwest A&F University, Yangling 712100, China

rticle history:eceived 27 June 2011eceived in revised form 13 March 2012ccepted 21 March 2012vailable online 31 March 2012

eywords:

a b s t r a c t

Tobacco viruses including Tobacco mosaic virus (TMV), Cucumber mosaic virus (CMV), Tobacco etch virus(TEV), Potato virus Y (PVY) and Tobacco vein banding mosaic virus (TVBMV) are major viruses infectingtobacco and can cause serious crop losses. A multiplex reverse transcription polymerase chain reactionassay was developed to detect simultaneously and differentiate all five viruses. The system used specificprimer sets for each virus producing five distinct fragments 237, 273, 347, 456 and 547 bp, representingTMV, CMV subgroup I, TEV, PVYO and TVBMV, respectively. These primers were used for detection of

obacco virusetectionultiplex PCR

T-PCR

the different viruses by single PCR and multiplex PCR and the results were confirmed by DNA sequenc-ing analysis. The protocol was used to detect viruses from different parts of China. The simultaneousand sensitive detection of different viruses using the multiplex PCR is more efficient and economicalthan other conventional methods for tobacco virus detection. This multiplex PCR provides a rapid andreliable method for the detection and identification of major tobacco viruses, and will be useful for

epidemiological studies.

. Introduction

In China, the principal viruses infecting tobacco crops areobacco mosaic virus (TMV), Cucumber mosaic virus (CMV), Tobaccotch virus (TEV), Potato virus Y (PVY) and Tobacco vein bandingosaic virus (TVBMV), which affect seriously the yield and qual-

ty of tobacco (Nicotiana tabacum) and cause great economic lossesWang et al., 2005; Zhang et al., 1998).

Sensitive, rapid and economical methods are essential fortudying these infections. Many methods, such as enzyme-linkedmmunosorbent assay (ELISA), polymerase chain reaction (PCR),ot-immunobinding assay (DIBA) and multiplex microsphere

mmunoassay (MIA) and reverse transcription loop-mediatedsothermal amplification (RT-LAMP) are able to detect differentiruses.

TMV, a member of the genus Tobamovirus, is the most com-on virus found in tobacco plants. ELISA (Van Regenmortel and

urckard, 1980), RT-LAMP (Liu et al., 2010), and oscillatory-ow reverse transcription-polymerase chain reaction (RT-PCR)

icrofluidics based on the capillary reactor (Wang et al., 2009) have

een developed for TMV detection.

∗ Corresponding author. Fax: +86 029 87092716.E-mail address: wuyf@nwsuaf.edu.cn (Y. Wu).

166-0934/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.jviromet.2012.03.029

© 2012 Elsevier B.V. All rights reserved.

CMV belongs to genus Cucumovirus, which is one of the mostcommon plant viruses. According to the serological relationshipand nucleic acid identity, CMV isolates were classified in two mainsubgroups named subgroup II and subgroup I (Palukaitis et al.,1992). Based on analysis of a large number of CP genes and 5′

nontranslated regions of CMV isolates’ RNA3, subgroup I was clas-sified in subgroups IA and IB (Roossinck et al., 1999). Serologicaldetection of CMV by ELISA and DIBA (Niimi et al., 1999; Zein andMiyatake, 2009), and molecular detection by RT-PCR and hybridiza-tion (Maoka et al., 2010a, b; Sugiyama et al., 2008) have beendeveloped.

TEV belongs to genus Potyvirus and causes serious economiclosses of tobacco plants. Current detection methods include sero-logical diagnosis by ELISA (Legnani et al., 1996), and moleculardiagnosis by RT-PCR (Lockhart et al., 2010).

PVY is a type member of the genus Potyvirus and is a single-stranded RNA virus occurring world wide. Various detectionmethods have been used for detecting of PVY. These include ELISA(Crosslin et al., 2005), MIA (Bergervoet et al., 2008), RT-LAMP (Nie,2005), non-radioactive nucleic acid hybridization (NASH) (Janczuret al., 2006), RT-PCR (Hogue et al., 2006; Xu and Nie, 2005), RT real-time PCR (Kogovsek et al., 2008) and hybridization (Hataya et al.,

1994; Maoka et al., 2010a, b).

TVBMV is a distinct species of the largest plant virus genusPotyvirus, family Potyviridae. It was first discovered in Taiwan in1964 (Chin, 1966). Symptoms of infected tobacco plants include

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hlorotic lesions on leaves, vein banding, and systemic necroticesions. Colloidal gold strip (Ji et al., 2009) and RT-PCR (Tian et al.,007) have been developed for TVBMV detection.

Multiplex PCR is a variant of PCR which enables simultaneousmplification of many targets of interest in one reaction by usingore than one pair of primers. It is more efficient and cost-effective

han other conventional methods. This technique has become aaluable tool for the detection of different viruses (Bertolini et al.,001; Dovas and Katis, 2003; Ito et al., 2002; Ragozzino et al., 2004;ga and Tsuda, 2005).

In this study, an improved multiplex PCR system was estab-ished which can detect simultaneously five tobacco viruses TMV,MV subgroup I, TEV, PVYO and TVBMV. This is the first report onetection of five tobacco viruses using multiplex PCR. It is capa-le of diagnosing mixed infections with tobacco viruses, which willllow the study of the epidemiology of virus infections in tobacco.

. Materials and methods

.1. Plant and viral materials

The tobacco viruses used in this study were TMV, CMV sub-roup I, TEV, PVYO and TVBMV. The tobacco plants (N. tabacum)ollected from the different tobacco fields of Shaanxi, Shandong,eilongjiang and Yunnan province were screened for TMV, CMV

ubgroup I, TEV, PVYO and TVBMV subgroup group III by RT-PCR.eaves collected from healthy tobacco and leaves infected withirus were stored at −80 ◦C for later use.

.2. RNA extraction and reverse transcription

Total RNA was extracted from healthy and infected leaf samplessing the Universal Plant Total RNA Extraction Kit (BioTeke) andhe first-strand cDNA was synthesized using Murine MLV-reverseranscriptase (Promega, Madison, WI, USA) according to the manu-acturer’s instructions. For cDNA synthesis, random primer (9mer)ather than oligo-dT was used because the TMV and CMV genomesre not polyadenylated.

.3. Primer design

To make the multiplex reaction work properly, all the primersere designed based on specificity and compatibility. Five pairs of

pecific primer were designed for single and multiplex PCR ampli-cation of conserved areas within the coat protein gene of TMV,MV subgroup I, TEV, PVYO and TVBMV. The nucleotide sequenceearch program provided by the National Center for Biotechnologynformation (NCBI) (http://www.ncbi.nlm.nih.gov/blast) was usedo obtain the coat protein gene sequences of each virus. Specificucleotide regions that were used for primer design were selected

rom sequence alignments generated by DNAMAN (Lynnon Biosoft,uebec, CA). All the primers were designed with similar anneal-

ng temperatures to facilitate multiplex PCR amplification. Theligonucleotide sequences of these primers are shown in Table 1.

.4. Single PCR

First stranded cDNA solution (2 �l) was added to a PCR tubeontained 2.4 �l of 25 mM MgCl2, 2.8 �l dNTP mixture (2.5 mMach), 2.5 �l of 10× PCR buffer and 0.5 �l of 5 U/�l Taq polymeraseTaKaRa Biotechnology) and 2 �l sense and antisense primers10 �M each) in a total volume of 25 �l. The samples were ampli-

ed using the DNAEngine Peltier Thermal Cycler (Bio-Rad, Hercules,A). The PCR protocol was 94 ◦C for 3 min, followed by 35 cycles of4 ◦C for 30 s, 48 ◦C for 30 s, 72 ◦C for 1 min, and a final extensiont 72 ◦C for 10 min. PCR products were analyzed by electrophoresis

Methods 183 (2012) 57– 62

on a 3% agarose gel, stained in ethidium bromide solution, and pho-tographed under UV illumination. Molecular sizes of the amplifiedfragments were determined by comparison with Marker1 (Dong-sheng Biotech, GuangZhou, China) as molecular weight marker.

2.5. Multiplex polymerase chain reaction

Multiplex PCR amplification was optimized to enable simultane-ous amplifications of all five targets in one reaction using five setsof virus-specific primers. In the multiplex PCR, a primer mixturecontaining 10 �M each of forward and reverse primer was made.The RT products were mixed in the equivalent amounts as multi-plex PCR template. The PCR conditions were optimized with a rangeof parameters including concentrations of DNA polymerase, primersets, MgCl2, and dNTPs. PCR cycle numbers, annealing temperature,and extension time were also optimized. Following optimization,PCR contained 2 �l cDNA, 2.5 �l of 25 mM MgCl2, 2.8 �l dNTP mix-ture (2.5 mM each), 2.5 �l of 10× Polymerase Buffer, 2 �l of 5 U/�lTaq polymerase (TaKaRa Biotechnology), and 2 �l of primer mix-ture (10 �M of each primer) in a total volume of 25 �l. The multiplexPCR was performed as follows: 35 cycles of denaturation at 94 ◦C for30 s, primer annealing at 51 ◦C for 30 s and then primer extensionsat 72 ◦C for 1 min with an initial denaturation at 94 ◦C for 3 min anda final extensions at 72 ◦C for 90 s. PCR products were analyzed byelectrophoresis on a 3% agarose gel.

2.6. Cloning and sequencing

In order to confirm the identity of the amplified products, themultiplex PCR products were purified from the agarose gels usingGel Extraction Kit (BioTeke). These fragments were ligated intothe pMD18-T simple vector, cloned into Escherichia coli JM109 andsequenced. Clones containing an insert were selected by colonyPCR and sequenced by Applied Biosystems 3730 DNA Analyzer(Applied Biosystems, USA) with M13 forward and reverse primers.These sequences were then verified by a BLAST search of the NCBInucleotide database (http://www.ncbi.nlm.nih.gov/blast).

2.7. Specificity testing of the five primer pairs

Specificity testing was performed for the positive controls ofTMV, CMV subgroup I, TEV, PVYO and TVBMV to determine thespecificity of the mixture of five primer pairs. Two combinationswere tested: (i) the PCR mixture containing five primer pairs andeach single template (cDNA), and (ii) five primer pairs and fivetemplates.

2.8. Sensitivity testing of the single PCR and multiplex PCR

Total RNA preparations from viral infected plants were adjustedto 100 ng/�l. To compare the sensitivity level of single and mul-tiplex PCR, 10-fold serial dilutions (100–10−5) in distilled watercontaining the five viral cDNA were tested by single and multiplexPCR. The single and multiplex PCR were carried out simultaneouslyfor the comparison of the dilution series.

2.9. Amplification in the presence of different amounts of targetviruses

To investigate whether the different viral concentrations couldinfluence the detection efficiency of this multiplex PCR assay, 10-

fold serial dilutions (100–10−4) in distilled water containing thefive viral cDNA were prepared. Two combinations were tested: (i)the PCR mixture containing five primer pairs and five templatescontaining equal volumes of cDNA from different dilutions, and (ii)

J. Dai et al. / Journal of Virological Methods 183 (2012) 57– 62 59

Table 1Virus-specific primers for multiplex PCR.

Target virus Primer Sequence 5′–3′ Tm (◦C) GC (%) Amplicon size (bp) NCBI accessiona

TMV TMVF TAGACCCGCTAGTCACAG 48.1 55.6 237 JN711115TMVR CAGAGGTCCAAACCAAAC 49.9 50.0

CMV CMVF GTGGGTGACAGTTCGTAAA 50.8 47.4 273 JN711116CMVR GTGGGAATGCGTTGGT 51.2 56.3

TEV TEVF TGATGGATGGTGAGGAG 47.6 52.9 347 JN711117TEVR GTGCCGTTCAGTGTCTT 47.6 52.9

PVY PVYF CCGAGAATCAAGGCTATC 49.8 50.0 456 JN711118PVYR CGCTAAACCTACATCCC 47.8 52.9

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in single PCR were 10 for TMV, CMV subgroup I, TEV, PVY andTVBMV (Fig. 2, Lane 5). The detection sensitivity of multiplex PCRdecreased slightly for CMV subgroup I, TEV and PVYO compared tosingle PCR.

TVBMV TVBMVF GAGGTCGTGAACTTACAGC

TVBMVR TTTCCTCCTTCGTGCT

a The coat protein genes of the five viruses were sequenced and submitted to NC

ve primer pairs and five templates containing equal volumes ofDNA from same dilutions.

.10. Validation the multiplex PCR result by ELISA

Tobacco leaf samples with symptoms collected from fields inhina were detected by multiplex PCR. The same samples wereest by ELISA to confirm the result of multiplex PCR. In ELISAssay, five kinds of antiserum (Neogen Corporation, USA) weresed to determine TMV, CMV subgroup I, TEV, PVYO and TVBMV,espectively. The antiserum of CMV is specific for CMV subgroup, and the antiserum of PVY is specific for O strain. Leaf tissue wasiluted 1:10 (w/v) in PBS-Tween buffer (pH 7.4) and ground. Thentiserum was diluted to 1:200 (v/v) in coating buffer (0.015 Ma2CO3, 0.035 M NaHCO3 and 0.003 M NaN3, pH 9.6) and incu-ated on microtitre plates at 37 ◦C. After two hours, the platesere washed in PBS-Tween and sap extract was added to theells. Plates were subsequently incubated at 37 ◦C for two hours

nd again washed in PBS-Tween. Alkaline phosphatase-conjugatedntiserum was diluted 1:200 in PBS-Tween (pH 7.4) and incu-ated at 37 ◦C for two hours. After a final washing step, substratep-nitrophenyl phosphate disodium) was added at 1 mg/ml in sub-trate buffer (1 M diethanolamine and 0.003 M NaN3, pH 9.8).eactions were measured at 405 nm with an ELISA reader (Bio-TekLx808) after incubation at room temperature for one hour. Sam-les were considered positive if optical density values exceeded theean background level by a factor three. Mean background levelsere determined for each ELISA plate by measuring at least twoells that contained all reagents except the sap extract.

. Results

.1. Optimization of the multiplex PCR

Various parameters were considered to optimize the multi-lex PCR for the mixed RNA targets from TMV, CMV subgroup I,EV, PVYO and TVBMV. Parameters such as annealing tempera-ure (42–57 ◦C), extension time (40 s–90 s) and number of cycles20–45) were evaluated using virus infected plants. For multiplexCR amplification, optimum results were obtained with a 48 or1 ◦C annealing temperature for 90 s using 35 cycles. Other param-ters such as concentrations of DNA polymerase, MgCl2, and dNTPsere also evaluated (results not shown).

.2. Specificity of the multiplex PCR

The specificity of the mixture of five primer pairs used in theresent study was tested and shown in Fig. 1. Each pair of primersmplified the viral genomes of positive controls and generated five

52.6 547 JN711119 50.0

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different sizes of amplicons of 237, 273, 347, 456, and 547 bp specif-ically and independently by TMVF and TMVR for TMV, CMVF andCMVR for CMV subgroup I, TEVF and TEVR for TEV, PVYF and PVYRfor PVYO and TVBMVF and TVBMVR for TVBMV. No cross-reactionwith non-targets was identified.

3.3. Confirmation of PCR specificity by DNA sequencing

To confirm the specificity of PCR products amplified by multi-plex PCR, all positive amplicons from multiplex PCR were clonedand sequenced separately. The sequences were compared with thedeposited GenBank sequences to confirm the specificity of PCR-amplified DNA fragments. Sequences of amplified products showedhigh identity to the coat protein gene sequences of TMV, CMV sub-group I, TEV, PVYO and TVBMV respectively.

3.4. Sensitivity testing of single PCR and multiplex PCR

The detection limits of the multiplex PCR were determined usinga 10-fold serial dilution of purified cDNA. For the 10-fold dilutionseries, the highest dilution at which multiplex PCR showed positiveresults was 10−4 for TMV and TVBMV and 10−3 for CMV subgroup I,TEV and PVYO, respectively (Fig. 2, Lanes 10 and 11). The detectionlimit of the single PCR was determined analogously as described forthe multiplex reaction. The positive results of the highest dilution

−4 O

Fig. 1. Specificity testing of the multiplex PCR assay with a mixture of five primerpairs for five positive controls. Lanes 1–5, TMV, CMV, TEV, PVY, and TVBMV, respec-tively; Lane 6, the mixture of TMV, CMV, TEV, PVY, and TVBMV positive controls; M,100 bp molecular marker.

60 J. Dai et al. / Journal of Virological Methods 183 (2012) 57– 62

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ig. 2. Comparison of the sensitivity between multiplex PCR and single PCR for the, negative control; Lanes 1–6, sensitivity testing of single PCR; lanes 7–12, sensitiv

.5. Effects of different amounts of target RNA to the sensitivity ofultiplex PCR

To evaluate the sensitivity of multiplex PCR, a mixture of differ-nt concentrations of the five viruses was amplified in a multiplexCR. Results are summarized in Table 2. The results showed thatetection of the five viruses was not influenced if the concentra-ion of the five viruses was similar. But the detection of the viruses

ith low concentration (TMV, 10−4; CMV subgroup I, 10−3; TVBMV,

0−4) were not detected if the concentration of one of other virusesas high (10◦).

able 2nterference test of the multiplex PCR.

Pathogens

TMV CMV TEV PVY TVBMV

100 10−4 10−3 10−2 10−1

10−1 100 10−4 10−3 10−2

10−2 10−1 100 10−4 10−3

10−3 10−2 10−1 100 10−4

10−4 10−3 10−2 10−1 100

100 100 100 100 100

10−1 10−1 10−1 10−1 10−1

10−2 10−2 10−2 10−2 10−2

10−3 10−3 10−3 10−3 10−3

10−4 10−4 10−4 10−4 10−4

, positive result for PCR test; −, negative result for PCR test. The concentration of total R

ld serial dilutions of positive controls. A, TMV; B, CMV; C, TEV; D, PVY; E, TVBMV;sting of multiplex PCR; M, 100 bp molecular marker.

3.6. Detection of tobacco virus from naturally infected samples bymultiplex PCR and ELISA

Tobacco leaf samples with symptoms collected from fields inChina were detected by multiplex PCR and ELISA. The results ofcomparisons between multiplex PCR and ELISA indicated that theassays give equivalent results in all but two samples which werepositive for TVBMV (sample 1) and TEV (sample 3) using the mul-

tiplex PCR but negative by ELISA (Table 3).

From 16 samples, most of these field samples showed differ-ent combinations of these five tobacco viruses (Fig. 3). Shaanxi

Multiplex PCR results

TMV CMV TEV PVY TVBMV

+ − + + ++ + − + ++ + + − ++ + + + −− − + + ++ + + + ++ + + + ++ + + + ++ + + + ++ − − − +

NA from viral infected plant was 100 ng/�l.

J. Dai et al. / Journal of Virological Methods 183 (2012) 57– 62 61

Table 3Results for field samples tested by ELISA and multiplex PCR.

Sample Location ELISA Multiplex PCR

TMV CMV-I TEV PVYO TVBMV TMV CMV-I TEV PVYO TVBMV

1 Shaanxi − + + + − − + + + +2 Shaanxi − + − + − − + − + −3 Shaanxi − + − + − − + + + −4 Shaanxi + + − + − + + − + −5 Shandong − + − − + − + − − +6 Shandong + − + − − + − + − −7 Shandong + − − + − + − − + −8 Shandong + − + − + + − + − +9 Heilongjiang − + − + − − + − + −10 Heilongjiang + − − − + + − − − +11 Heilongjiang + − − + − + − − + −12 Heilongjiang + + + − − + + + − −13 Yunnan − − + − + − − + − +14 Yunnan + + − + − + + − + −15 Yunnan − + − + − − + − + −16 Yunnan + − − + − + − − + −

+

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, positive result for test; −, negative result for test.

amples looked more dominantly infected by CMV subgroup I andVYO than TMV, TEV and TVBMV (Fig. 3A, Lanes 2–5) as indicatedy the number of the bands. The plant samples from Shandonghowed multiple infections with the presence of CMV subgroup Ind TVBMV (Fig. 3A, Lane 6), TMV and TEV (Fig. 3A, Lane 7), TMV andVYO (Fig. 3A, Lane 8) and TMV, TEV and TVBMV (Fig. 3A, Lane 9).eilongjiang samples showed a different trend with the presencef CMV subgroup I and PVYO (Fig. 3B, Lane 2), TMV and TVBMVFig. 3B, Lane 3), TMV and PVYO (Fig. 3B, Lane 4) and TMV, CMVubgroup I and TEV (Fig. 3B, Lane 5). Field samples from Yunnanhowed multiple infections with the presence of TEV and TVBMVFig. 3B, Lane 6), TMV, CMV subgroup I and PVYO (Fig. 3B, Lane), CMV subgroup I and PVYO (Fig. 3B, Lane 8) and TMV and PVYO

Fig. 3B, Lane 9). Following gel electrophoresis, the bands of PCRroducts were clear and there were no discernible differences inhe size of the virus amplicons obtained from the different fieldamples indicating that the multiplex PCR assay could be used toetect the five tobacco viruses from a wide range of field areas.

ig. 3. Simultaneous detection of multiple tobacco viruses from field plant samplessing multiplex PCR. Field samples from different areas of China: Shaanxi (panel, Lanes 1–4), Shandong (panel A, Lanes 5–8), Heilongjiang (panel B, Lanes 9–12),unnan (panel B, Lanes 13–16). N, healthy control; P, positive control of all fiveiruses simultaneously amplified with multiple primer pairs; M, 100 bp moleculararker.

4. Discussion

In order to diagnose tobacco viruses, it is essential to developa rapid and reliable method to detect them. This study devel-oped an multiplex PCR method for rapid detection of five tobaccoviruses (TMV, CMV subgroup I, TEV, PVYO and TVBMV) using spe-cific primer sets for each virus.

In this study, the multiplex PCR method was optimized against arange of parameters including concentrations of DNA polymerase,primer pairs, MgCl2, and dNTPs, annealing temperature, extensiontime and cycle numbers. The sensitivity of multiplex PCR decreasedslightly for CMV subgroup I, TEV and PVYO compared to single PCRwhen using the 10-fold dilution series of templates (Fig. 2). Thereason for this decrease may be that in the multiplex assay thecocktail primers compete for all the five viral templates rather thanfor one and therefore the detection end point is lower (Uga andTsuda, 2005).

ELISA, one of the conventional detection methods, has been acost-efficient method to detect a large number of virus-infectedsamples (French, 1995). However, this technique has the disad-vantage of being less reliable for detection of virus with low virustiter (Canning et al., 1996). Additionally, identification of differentviruses by ELISA requires different antisera for each virus. Cur-rently, RT-PCR provides a molecular method and is considered tobe a reliable and sensitive method to detect and differentiate dif-ferent pathogens. In addition, with its faster testing procedure, andreagent savings against single RT-PCR, multiplex PCR assay has pro-gressed to become a convenient, rapid, and cost-effective tool foridentification of different viruses. It has been applied in many areasof pathogen detection, since it was first described by Chamberlainet al. (1988). There are several examples using multiplex PCR whichwere successful in amplifying a range of plant viruses (Bariana et al.,1994; Bertolini et al., 2001; Deb and Anderson, 2008; Nie and Singh,2000; Okuda and Hanada, 2001). However, this is the first time thatmultiplex PCR was used to detect five viruses in tobacco.

The method of nucleic acid extraction is of great importanceto the results obtained using multiplex PCR. Various nucleicacid extraction methods for multiplex PCR have been evaluated(Bertolini et al., 2001; Roy et al., 2005; Thompson et al., 2003). In thisstudy, the Universal Plant Total RNA Extraction Kit (BioTeke) can

effectively separate RNA with polysaccharides and remove easilypolyphenols, so it was suitable due to its simplicity, reproducibilityand speed for the RNA extraction of plants with polysaccharidesand polyphenols such as tobacco.

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In order to make the multiplex PCR assay act as a universaliagnostic tool, it is important to design specific primers for eachirus. The information on the genetic diversity of PVY and TVBMVHu et al., 2009; Li et al., 2006; Tian et al., 2007, 2011) and detec-ion of TMV, CMV and TEV (Bhat and Siju, 2007; Lockhart et al.,010; Sliwa et al., 2008; Wang et al., 2009) helped to design specificrimers that have a wide geographical range. Additionally, param-ters such as the extent of homology with the targets, expectedmplified fragment size, primer length and annealing temperaturesere considered.

In conclusion, a new multiplex PCR assay was developed toetect TMV, CMV subgroup I, TEV, PVYO and TVBMV from infectedobacco plants. The multiplex PCR assay developed here is a rapid,eliable, sensitive and cost-effective diagnostic method. It canimultaneously detect these five tobacco viruses in infected leafissue across a wide range of field samples in one single reaction. Itould be useful for the control of tobacco viruses and for the stud-

es of the distribution, characterization and epidemiology of theseve tobacco viruses.

cknowledgments

The study was supported by the Key Technology Program ofhina National Tobacco Corporation (110200902046) and the 111roject from the Education Ministry of China (No. B07049).

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