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Journal of Virological Methods 183 (2012) 215– 218

Contents lists available at SciVerse ScienceDirect

Journal of Virological Methods

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

hort communication

highly sensitive single-tube nested PCR assay for the detection of Pineappleealybug wilt associated virus-2 (PMWaV-2)

.K. Deya, Hong Linb, W.B. Bortha, M.J. Melzera, J.S. Hua,∗

Department of Plant and Environmental Protection Sciences, University of Hawaii at Manoa, 3190 Maile Way, Honolulu, HI 96822, USACrop Diseases, Pests and Genetics Research Unit, San Joaquin Valley Agricultural Science Center, USDA-ARS, 9611 S. Riverbend Ave, Parlier, CA 93648, USA

rticle history:eceived 24 September 2011eceived in revised form 15 March 2012ccepted 21 March 2012vailable online 30 March 2012

eywords:

a b s t r a c t

An assay was developed for the detection of Pineapple mealybug wilt associated virus-2 (PMWaV-2), animportant factor in the etiology of mealybug wilt of pineapple. The assay combines reverse transcriptionof RNA isolated from pineapple with a specific and very sensitive, single, closed-tube nested polymerasechain reaction (PCR) to amplify a segment of the coat protein gene of the PMWaV-2. The outer primerswere designed to anneal at higher temperatures than the nested primers to prevent primer competitionin consecutive amplification reactions. To reduce potential competition further, the outer primers were

MWaV-2TDP-TaqMan®qPCRested PCRT-PCRmpeloviruslant viruses

used at one-thousandth the concentration of the nested primers. The specificity and sensitivity of thisassay are much greater than PCR using only a single primer-pair. A TaqMan® probe was also designedfor use in quantitative PCR to detect and quantify the PCR amplification products directly in a single-tube assay. The advantages of the single-tube assays using both conventional and quantitative PCR arereduced handling time and prevention of cross contamination compared to regular nested PCR in whichthe reactions are carried out in two separate tubes.

The pineapple mealy bug wilt associated viruses (PMWaVs)re a complex of viruses in the genus Ampelovirus, family Clos-eroviridae that infect pineapple (Ananus comosus) (Melzer et al.,001, 2008; Sether et al., 2005, 2009; Gambley et al., 2008). Cur-ently there are five known PMWaVs infecting pineapple in Hawaii,uba, and Australia (Gambley et al., 2008). In Hawaii the presencef PMWaV-2 and the feeding of pineapple mealybug (Dysmicoc-us spp.) was shown to be necessary for symptom developmentSether and Hu, 2002; Sether et al., 1998) whereas in Australiao such clear association has been found (Gambley et al., 2008).arious methods have been developed for detection of PMWaVs,

ncluding ELISA, tissue blot immunoassay (TBIA), dsRNA analysis,nd conventional reverse transcription (RT) PCR. (Gunasinghe anderman, 1989; Hu et al., 1993, 1997; Walkman et al., 1995; Sethert al., 2001, 2005). Using a quantitative (q) RT-PCR assay, Subere2009) found the titer of PMWaV-2 in different pineapple tissueso be highly variable (Subere 2009, PhD dissertation, University ofawaii). Although qRT-PCR is extremely rapid, specific, and sen-

itive, the cost of the instrument and chemicals is prohibitive forany laboratories. Another very sensitive technique is nested PCR,

hich does not involve costly equipment or chemicals. However,

ecause it often involves the transfer of PCR amplification productsrom one reaction tube to another, the potential for contamination

∗ Corresponding author. Tel.: +1 808 956 7281; fax: +1 808 956 2832.E-mail address: johnhu@hawaii.edu (J.S. Hu).

166-0934/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.jviromet.2012.03.025

© 2012 Elsevier B.V. All rights reserved.

is high. Llop et al. (2000) took advantage of the sensitivity of nestedPCR and eliminated the transfer of products from the first reactiontube to the second nested reaction tube by conducting both reac-tions in a single tube, thus reducing the potential for false positiveresults. This resulted in a very sensitive, reliable assay, which wasalso very easy to perform. Using this technique, Erwinia amylovorawas detected in plants prior to the onset of disease symptoms (Llopet al., 2000). To make this type of assay quantifiable, Lin et al.(2010) included a TaqMan® probe in the single-tube nested PCRwhich allowed them to detect Candidatus Liberibacter asiaticus, thebacterium associated with Huanglongbing (HLB), in asymptomaticcitrus plants. A highly sensitive nested RT-PCR assay in a singleclosed tube is described for detection of a plant RNA virus that maybe applicable for the detection of other plant viruses that may occurat very low titers in host tissues.

Two nested PCR primer sets (inner and outer) and a TaqMan®

probe were designed to amplify a portion of the 904 nt coat proteingene of PMWaV-2 using Beacon® Design Software v 7.0 (PremierBiosoft International, CA, USA) with the following criteria: GC%40–50, Tm = 55 + −2 ◦C, primer length = 18–22 bp, and amplicon sizeof 120–200 bp. The melting temperature (Tm) for the TaqMan®

probe was set 10 ◦C higher than the Tm for the inner primer pairs.To ensure high amplification efficiency, the primers and probes

were targeted to a region with no predicted secondary struc-tures. The same criteria were applied to design the outer primers,except that a Tm of 65 ◦C (10 ◦C higher than inner primer Tm)and longer amplicon size (300–500 bp) were selected. The selected

2 ogical Methods 183 (2012) 215– 218

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Fig. 1. Optimization of annealing temperature for inner nested primers from 54.5 ◦Cto 72.4 ◦C (top) and 65 ◦C to 75 ◦C for outer primers (bottom) in gradient PCR withcloned plasmid DNA of PMWaV-2 coat protein as a template. The strongest bandat the highest temperature selected was 55 ◦C for inner primers and 68 ◦C for outer

16 K.K. Dey et al. / Journal of Virol

rimers which met these criteria were the outer primer-forwardLinO-F) 5′-GTATAGGAGAGGCTCTGAAGGAACATTGC-3′ and outerrimer-reverse (LinO-R) 5′-CATCACGGAGTACCCAAGCAGTTCTTT-3′.he nested primers were inner primer forward (LinI-F) 5′-CATTTGATTCAACTCGCTATTACG-3′ and inner primer reverse (LinI-) 5′-GGGTACGAAAACCCGTTTAGGC-3′.

The TaqMan® probe (5′-GAGTGAGGTGGTTGAGAGAGCGGCGA-3′)as labeled at the 5′-end with 6-carboxyl-fluorescein (FAM) and

he 3-end was labeled with a quencher. The probe was synthesizedy Applied Biosystems (ABI, Foster City, CA, USA).

To determine the optimum annealing temperature for the outernd inner primers and the maximum range at which both therimers can operate, gradient PCR was conducted with anneal-

ng temperatures from 54.5 ◦C to 72.4 ◦C for the inner primer pairsnd 65–75 ◦C for outer primer pairs. Reactions were performed inolumes of 20 �l containing the PMWaV-2 coat protein (CP) geneloned in plasmid pGEM®-Teasy (Promega, Madison, WI, USA), theuter or inner primers and ImmoMix® (Bioline, Randolph, MA,SA). The reaction conditions were initial denaturation step at 95 ◦C

or 10 min followed by 35 cycles of 95 ◦C for 30 s, 45 s for anneal-ng at various temperatures, extension at 72 ◦C for 45 s, followed bynal extension at 72 ◦C for 7 min.

After primer-annealing temperatures were optimized, the opti-al inner and outer primer concentrations were determined.

rimer amounts tested were 0.05, 0.1, 0.5 or 1.0 pmol for the outerrimers, and 5 or 10 pmol for inner primers in a 20 �l PCR using

mmoMix® (Bioline) with reaction parameters of 95 ◦C for 10 min,ollowed by 20 cycles of 95 ◦C for 30 s, 68 ◦C for 45 s, and 72 ◦C for5 s. This initial PCR was followed by a denaturation step of 4 mint 95 ◦C and 35 cycles at 95 ◦C for 30 s, 55 ◦C for 45 s, and 72 ◦C for5 s and a final extension at 72 ◦C for 7 min. This PCR protocol iseferred as single closed tube dual primer-PCR (STDP-PCR).

The sensitivity of the STDP-PCR assay was tested and com-ared with conventional single primer pair PCR with only the

nner primers of the nested primer sets using cDNA as tem-late. The cDNA was synthesized with MMLV-reverse transcriptasePromega) using RNA extracted from PMWaV-2 infected pineapplelants with an RNeasy® Plant Mini Kit (Qiagen, Valencia, CA, USA).his cDNA was serially diluted in either water or genomic DNAxtracted from the lower white basal portions of PMWaV-2 freeineapple leaves using the DNeasy® Plant Mini Kit (Qiagen) beforeeing used as a template. For conventional single primer pair PCR,he primer amounts were 10 pmol each. The PCR conditions were5 ◦C for 10 min followed by 35 cycles of 95 ◦C for 30 s, 55 ◦C for5 s, and 72 ◦C for 45 s, and a 7 min extension at 72 ◦C. For STDP-CR, 0.1 pmol outer and 10 pmol inner primers were used with theCR conditions described above. Water was used as negative con-rol in all experiments and all PCR products were visualized by UVight in 1.2% (w/v) agarose gels stained with ethidium bromide.

To quantify the sensitivity of the assay, a Taqman® probe wassed in the STDP-PCR assay (STDP-qPCR) with 0.1 pmol outerrimers, 20 pmol of inner primers and 5 pmol of Taqman® probe. Toest the sensitivity of STDP-qPCR, in vitro generated RNA transcriptsynthesized from SpeI digested plasmid using MEGAscript®-T7ranscription kit (Ambion, Foster City, CA, USA) was used. RNAsere recovered and quantified on a ND-1000 spectrophotometer

Nanodrop, Thermo Scientific, USA). Aliquots were also visualizedy UV light in ethidium bromide stained 1% agarose gels to deter-ine the RNA quality. RNAs were then diluted to 100 ng aliquots

nd stored at −80 ◦C. Conversion of micrograms of single-strandedNA to picomoles and numbers of RNA molecules was performeds described by Olmos et al. (2005). These RNA transcripts were

iluted serially in water or genomic DNA extracted from the lowerhite basal portions of PMWaV-2 free pineapple leaves usingNeasy® Plant Mini Kit (Qiagen) at 10-fold intervals from 1.0 × 1010

opies/�l to one copy/�l of RNA and were converted into cDNA

primers, a difference of more than 10 ◦C from outer primers to prevent competitionof inner primers with outer primers at two different PCR cycles. M is molecularweight ladder and NC is water only control.

using 2 units MMLV reverse transcriptase (Promega) and 10 �gof random hexamer primers. The RNA transcripts were denaturedfor 5 min at 95 ◦C, chilled on ice, and cDNAs were synthesized at42 ◦C for 45 min followed by 95 ◦C for 5 min. After the reverse tran-scription reaction, 1 �l of resulting cDNA was used to comparethe sensitivity of STDP-qPCR to that of standard single-primer pairqPCR with only the inner primers of the nested primer sets, or wereused as standard curves to validate the assay using STDP-qPCR.For STDP-qPCR reactions, Sensimix® qPCR master mix (Bioline),0.1 pmol of each outer primer, 20 pmol of inner primers, and 5 pmolof TaqMan® probe in a 20 �l final reaction volume were used. Thecycling conditions were the same as described above for STDP-PCR. Fluorescence was detected at the end of each inner primerpair annealing step. Reactions were carried out in an IQ5 instru-ment (Bio-Rad, CA, USA). For single-primer pair qPCR amplification,20 pmol of primers, 5 pmol of TaqMan® probe with Sensimix® PCRmaster mix (Bioline) were mixed in a 20 �l final reaction volume.The conditions were 10 min at 95 ◦C followed by 35 cycles of 95 ◦Cfor 30 s, 55 ◦C for 45 s, and 72 ◦C for 45 s, with 7 min final extension.

To validate the STDP-qPCR assay using plant samples and com-pare it with standard single-primer pair qPCR, cDNA (1 �l) derivedfrom 10-fold dilutions (95 ng to 0.95 pg) of total RNA isolated froma PMWaV-2-infected or a PMWaV-2-free plants was used as tem-plate for both assays using the conditions described above. Tovalidate the assay further, and compare its sensitivity to other tech-niques, 10 field grown pineapple plants were tested for PMWaV-2by TBIA (Hu et al., 1997), RT-PCR (Sether et al., 1998), and the STDP-qPCR assay.

The outer primers were found to have an optimal annealingtemperature of 68 ◦C and generated a 350 bp product. The innerprimers had an optimal annealing temperature of 55 ◦C and gener-ated a 164 bp product. Ideally the inner primers should not amplifyany product during the first PCR at 68 ◦C. However, at this temper-ature the inner primers did produce some amplification products(Fig. 1, top). Increasing the annealing temperature by one degreeeliminated amplification by the inner primers, but the amplificationefficiency with the outer primers was slightly reduced, whereas at68 ◦C the sensitivity of the nested PCR assay was not found to becompromised (results not shown).

To minimize competition of the outer primers with the innerprimers during nested PCR, limiting amounts of outer primers weretested. For a 20 �L reaction, 0.1, 0.5 and 1.0 pmol of each outerprimer were combined with 5 or 10 pmol of each inner primer. Ofthe various combinations, 0.1 pmol of each outer primer, regardlessof inner primer concentration, was found to be the most sensitive(data not shown). Using 0.1 pmol of each outer primer, 10 pmolof each inner primer, and serially diluted cDNA from a PMWaV-2infected plant as template, STDP-PCR was found to be much moresensitive than PCR with inner primers alone (Fig. 2). To evaluate

the sensitivity and the amplification efficiency of STDP-qPCR withsingle-primer pair qPCR, RNA transcripts of the cloned PMWaV-2CP gene, serially diluted (representing 107 to 101 copies) in water orDNA isolated from PMVaV-2-free plants, were converted to cDNA.

K.K. Dey et al. / Journal of Virological

Fig. 2. Comparing sensitivity of STDP-PCR (top) to single-primer pair PCR (bottom)with cDNA as template amplified from PMWaV-2 infected plant RNA and diluted in9tl

TtewmtuodwPawPdq1tro

Fpr

-fold water or PMWaV-2 free genomic DNA (1–9). The numbers above lanes refero dilutions. NC is the water only negative control and M is the molecular weightadder.

he lowest detectable cDNA template for STDP-qPCR was foundo be 107 copies with a Ct value of 6.34 (Fig. 3, top). The earli-st that a product could be detected using single-primer pair qPCRas at 25 cycles with a Ct value of 21.81 (Fig. 3, bottom), which isuch less sensitive than STDP-qPCR. To validate the assays further,

otal RNA from PMWaV-2 infected plants was serially diluted andsed as template to test the sensitivity of STDP-qPCR for detectionf target RNA compared with single-primer pair qPCR. STDP-qPCRetected the PMWaV-2 CP sequence from 0.95 pg RNA (Fig. 4, top)hereas single primer amplification was only capable of detecting

CR products from 0.95 ng RNA input (Fig. 4, bottom). In a sep-rate experiment, 10 individual plants that tested inconclusivelyith tissue blot Immunoassay (TBIA) and that were negative for

MWaV-2 in RT-PCR with PMWaV-2 specific primers previouslyeveloped (Sether et al., 1998), were analyzed using the STDP-PCR assay. Surprisingly, 9 of 10 plants with average Ct value of

3.53 ± SD 3.04 were found to be positive in STDP-qPCR. The virusiter was quantified as 1.56 pg in one sample (1.56 pg to 6.37 pgange) using the STDP-qPCR assay. This is below the detection limitf either single primer PCR or serological methods.

ig. 3. Comparison of sensitivity of STDP-qPCR (top) with 0.1 pmol outer primers, 20 pmair qPCR with 20 pmol inner nested primers and 5 pmol TaqMan® probe (bottom). The

eaction.

Methods 183 (2012) 215– 218 217

Pineapple is propagated vegetatively through crowns, slips, andsuckers. Infected planting materials are often symptomless andthus are disseminated widely in newly planted fields. These fac-tors underscore the need for a rapid and reliable detection assayfor large scale screening of PMWaV-2 in pineapple plants. NestedPCR is more sensitive than quantitative PCR but has always beenproblematic because of the potential for false positives arising dur-ing manipulation of PCR products. To overcome this drawback,Llop et al. (2000) modified the nested PCR for detection of Erwiniaamylovora by conducting nested PCRs in a single tube, and carefullyadjusting annealing temperatures of the outer and inner primersto overcome potential cross-contamination problems. They wereable to show with great reliability that asymptomatic plants wereinfected by the bacterial pathogen Erwinia amylovora. The causativeagent of the citrus disease, Huanglongbing (HLB) is caused byCandidatus Liberibacter sp. where the pathogen is often asymp-tomatic in infected trees for long periods. Candidatus Liberibactersp. is unculturable and occurs in very low and uneven titers inhost plants. Using quantitative nested PCR, 12% more HLB-positive,field-collected citrus samples were detected compared to usingonly conventional PCR (Lin et al., 2010). To our knowledge this isthe first study using a similar technique to detect plant viruses.It was shown that STDP-PCR used with or without a TaqMan®

probe, can be a useful detection system. For the routine diagnosisof difficult to detect plant viruses STDP-PCR without the TaqMan®

probe can be used. However, where qPCR is available and quan-

tification is desired, STDP-qPCR can be used. The usefulness of theassay becomes clear when pineapple plants that were healthy inappearance and that tested negative using conventional PCR orother techniques were shown to be infected by PMWaVs using the

ol inner primers and 5 pmol Taqman® probe in 20 �l reaction with single-primeramplification plot represents a dilution series of standards from 107 to 101 copies

218 K.K. Dey et al. / Journal of Virological Methods 183 (2012) 215– 218

F V-2 inc R (tops

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ig. 4. STDP-qPCR detection of PMWaV-2 from total RNA extracted from PMWaontaining total RNA at 95 ng, 9.5 ng, 0.95 ng, 95 pg, 9.5 pg, 0.95 pg using STDP-qPCignal.

TDP-qPCR assay described here. Using similar assays, the viruseshat cause many important viral diseases such as citrus tristeza andrapevine leaf roll may be detected very early in non-symptomaticissues resulting in more efficient control strategies for disease

anagement.

cknowledgement

The research project was funded in part, by a grant through theropical/Subtropical Agricultural Research Program (Project No.AW00985-10145).

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