Multiplex PCR is an impoassay. Previous research hprimer design, PCR compoprimers to form complexnovel form of primer secotant factors affecting succinhibitors were tested, inamplification; decrease oaddition of bovine serumparable to the simplex PCvirus, Strawberry latent rinwas also included. Theseprotocols for the detectionthe environment.
1. Introduction
Global agriculture is largely dependent on crops which aregrown in areas outside their natural range and therefore interna-tional movement of germplasm is vital. To ensure that damagingpests and diseases are not moved inadvertently with plantgermplasm, reliable, rapid and inexpensive procedures are requiredfor detecting pests during importation or local surveillance pro-grams. Serological and molecular assays including enzyme-linkedimmunosorbent assay (ELISA) and polymerase chain reaction (PCR)have been developed for the detection of viruses (Narayanasamy,1997; Stevens et al., 1997; Thomson and Dietzgen, 1995). How-ever, the application of these assays in quarantine programmes hasinherent problems, including a lack of desirable sensitivity, exces-sive costs and/or they are time consuming. Multiplex PCR enablesthe advantages of ELISA and PCR, in terms of sensitivity, processingcost and time, to be combined and therefore provides significantadvantages in pathogen testing programmes (Elnifro et al., 2000).
technique for detecting a variety of pathogens simultaneously in a singleused on optimising the factors affecting reliable multiplex PCR, including
s and conditions, and inhibitors in samples. In this study, the interaction ofdary structures including visible dimers and invisible “primer clusters”, astructure found during this research, were shown to be the most impor-
l multiplex PCR. Approaches to mitigate primer interaction and eliminateg: reduction of primer concentrations especially those with preferentialextension temperature; increase of extension time and PCR cycles; andin. Based on these approaches, a multiplex RT-PCR with sensitivity com-
individual viruses was developed for the detection of Raspberry ringspotvirus and Tomato bushy stunt virus. A plant internal amplification controlaches may be useful as a guideline for the development of multiplex PCRher pathogens or organisms associated with plants, humans, animals and
Although, multiplex PCR assays have been developed for detect-ing viral infections in strawberry and other plants (Menzel et al.,
2002; Roy et al., 2005; Sanchez-Navarro et al., 2005), the develop-ment of a multiplex PCR is tedious and time consuming. Problemssuch as poor sensitivity for one or more targets can be encoun-tered, especially when the concentrations of the templates presentin the reaction are different. The mechanisms leading to these prob-lems are complicated and have been attributed to PCR drift andselection (Polz and Cavanaugh, 1998; Wagner et al., 1994), primerdimer formation (Brownie et al., 1997), and mispriming (Puskas andBottka, 1995; Shigemori et al., 2005). Primer interaction can leadto primer dimer formation (Li et al., 2007; Rychlik, 1995), primerhairpin formation (Li et al., 2007) and primer–template mispriming(Puskas and Bottka, 1995). Strategies have been proposed to elim-inate primer interaction by the careful selection of primer pairs,hot-start PCR, and optimisation of PCR components (Elnifro et al.,2000). However, difficulty still exists in the development of a prac-tical approach to solve these problems, because it is not easy todesign completely compatible primers. Also, the means to predictthe performance of primer pairs relies on a diversity of softwarewhich produces variable outputs.
Some plant-derived compounds such as polysaccharides,polyphenolics, pectin and xylan, may be co-extracted with nucleic
T. Wei et al. / Journal of Virological Methods 151 (2008) 132–139 133
Table 1Published primers used for development of the multiplex PCR in this study
acid and inhibit PCR (Demeke and Adams, 1992; Malvick andGrunden, 2005; Wilson, 1997) which makes the development ofmultiplex PCR more complicated. For example, plants such asstrawberry are rich in a range of phenolics and polysaccharideswhich make it difficult to extract nucleic acid of amplifiable qual-ity (Porebski et al., 1997; Seeram et al., 2006; Thompson et al.,2003). Dilution of the templates, addition of specific reagents to theextraction buffer or PCR mixture, or immunocapture are required toeliminate PCR inhibitors and thus improve PCR amplification effi-ciency (Koonjul et al., 1999; Singh et al., 2002; Vigano and Stevens,2007; Wilson, 1997).
Strawberry is an important crop grown worldwide and isaffected by a variety of viruses which can cause significant losses(Barritt and Loo, 1973; Martin and Tzanetakis, 2006). In thisstudy, three viruses infecting strawberry Raspberry ringspot virus(RpRSV) (Cadman, 1956), Strawberry latent ringspot virus (SLRSV)(Lister, 1964), and Tomato bushy stunt virus (TBSV) (Kaitasowa andKondakowa, 1989) together with a plant internal amplificationcontrol (PIAC) were used as targets to demonstrate that primerinteraction is a crucial factor affecting the development of reli-
able multiplex PCR. In addition to the previously recognized primerdimers, primer self-annealing and self-looping, we postulate theexistence of a novel form of interaction between primers with-out any predictable secondary structure. We also propose newstrategies which aim to mitigate primer interaction and eliminateinhibitors from plant samples which resulted in a sensitive multi-plex PCR for the detection of RpRSV, SLRSV and TBSV in strawberrywith a PIAC. These strategies facilitate the development of multi-plex PCR by directly using published primers thereby eliminatingthe need to design novel PCR primers and protocols.
2. Materials and methods
2.1. Plant materials
Dried plant tissues (Nicotiana spp.) infected with RpRSV (Tarvitisolate) and TBSV (PV0285, Denmark isolate) were obtained fromthe Scottish Crop Research Institute, Dundee, United Kingdom, andDSMZ, Braunschweig, Germany, respectively. Blackberry (Rubus sp.)infected with SLRSV and healthy strawberry (Fragaria × ananassa)
were obtained from New Zealand. The identity of the three viruseswas confirmed by sequencing (data not shown).
2.2. Primers and analyses
The primers used in this study were designed origi-nally for the individual detection of viruses by conventionalPCR (Table 1). A pair of primers (Nad5-S and Nad5-AS)was also used for amplification of PIAC. The characteristicsof the primers (melting temperature (Tm), GC content, self-annealing, self-looping and annealing between primer pairs)were analyzed using Oligo Analyzer 1.0.2 (http://molbiol-tools.ca/molecular biology freeware.html) and used to predictpotential interactions and the compatibility of these primers formultiplex PCR. The homology between primer pairs was also ana-lyzed by DNAstar (Window 3.10a) to assist in the prediction ofpotential non-specific amplification of these primers in multiplexPCR.
2.3. RNA extraction and cDNA synthesis
Total RNA was extracted from infected plants and healthy straw-berry plants using the RNeasy® Plant Mini Kit (Qiagen) followingthe manufacturer’s protocol. The cDNA was synthesized usingSuperScriptII Reverse Transcriptase (Invitrogen) and random hex-amer primers (Invitrogen) in a total volume of 10 �l. As the threeviruses were not extracted from strawberry, RNA of each viruswas either used for cDNA synthesis directly or mixed with healthystrawberry RNA for comparison. The synthesized cDNA was storedat −20 ◦C.
2.4. PCR and effect of bovine serum albumin (BSA) in PCR
A preliminary evaluation of several Taq DNA polymerasesshowed that GoTaq® Green Master Mix (Promega) provide the bestamplification efficiency and was subsequently used in this study(data not shown). PCR assays were carried out in a total volumeof 10 �l containing 5 �l of 2× GoTaq® Green Master Mix, 1 �l ofprimers at specific concentrations, 1 �l of cDNA sample and thefinal volume was made up with nuclease-free water and an opti-
mum concentration of BSA. The entire volume of the PCR productswas analysed by electrophoresis on 2% agarose-TAE gels containingSYBR SafeTM gel stain (Invitrogen).
BSA (RIA grade, A7888-50G, Sigma–Aldrich, Auckland, NewZealand) at a final concentration ranging from 0.01 to 10 �g/�l(0.01–1%) was added to the PCR mixture to investigate its effecton PCR efficiency.
2.5. Impact of primer interaction on PCR efficiency andmitigation of interaction in multiplex PCR
Primer interactions were evaluated with individual and mul-tiple primer pairs at different concentrations (ranging from 62.5to 1660 nM/primer) on single and multiple cDNA templates insimplex or multiplex PCR assays. Primer interactions were alsoinvestigated using different PCR conditions including: (1) differentannealing times (20 s to 3 min), (2) different extension tempera-tures (60–72 ◦C), and (3) different extension periods (30 s to 3 min).The optimal PCR conditions to mitigate the primer interaction andimprove the amplification efficiency for all the investigated virustargets were selected for multiplex PCR.
2.6. Sensitivity and applicability of the developed multiplex PCR
The sensitivity of the developed multiplex PCR was evaluatedusing viral cDNA serially diluted in healthy strawberry cDNA andwas compared to simplex PCR done using the published conditions(Harris et al., 2006; Ochoa-Corona et al., 2006; Postman et al., 2004).Template sets containing a combination of viral cDNA at differ-
ent concentrations were constructed to mimic natural infections(i.e. containing one, two or all three viruses in different concentra-tions) and these were used to assess the potential application of themultiplex PCR in the field.
3. Results
3.1. Analysis of primers
The Tm of the primers ranged from 58 to 70 ◦C and the work-ing annealing temperatures for the four targets ranged from 50 to62 ◦C (Tables 1 and 2) suggesting that a common annealing temper-ature was possible between 50 and 58 ◦C. Preliminary experimentson the optimization of annealing temperature confirmed the pre-diction and an annealing temperature at 56 ◦C provided the bestamplification efficiency for the four investigated targets (data notshown). The PCR products amplified by each set of primers (Table 1)could be easily distinguished by electrophoresis.
Most of the primers had no self-annealing and/or self-loopingexcept three primers (SLRSV-F, SLRSV-R and Nad5-AS) which had aself-annealing energy between −0.95 and −6.68 kcal/mol. The two
Table 2Melting temperature (Tm), guanine/cytosine (GC) content and secondary structureof each primer predicted by Oligo Analyzer software
a Self-annealing means the annealing between different strands of single primer.
ethods 151 (2008) 132–139
primers (SLRSV-F and Nad5-AS) had a self-looping energy of 0.16and −1.69 kcal/mol (Table 2), respectively. There was no anneal-ing free energy for the forward and reverse primer pairs for RpRSVand TBSV, while the annealing for the primer pairs of SLRSV andPIAC were −2.8 and −3.76 kcal/mol, respectively (Table 3). Thefree energies of annealing between any two primers from differentprimer pairs ranged from 0 to −6.69 kcal/mol (Table 3). Notably, theprimers for SLRSV and PIAC had an annealing energy with all otherprimers, suggesting potential interactions between these primersmight cause problems in multiplex PCR.
The limited homology (15.8–46.7%) between different primers(Table 3) indicated that non-specific amplification of the targetswas unlikely because at least 75–80% homology between primerand target is considered necessary to cause cross-hybridization(Kane et al., 2000).
3.2. Effects of BSA
Using undiluted cDNA, amplification was only achieved forRpRSV and TBSV from the original hosts, but not for SLRSV fromblackberry or RpRSV, SLRSV and TBSV spiked into healthy straw-berry RNA before reverse transcription (RT) (data not shown).However, all targets were amplified when BSA was added to thePCR. It was found that small concentrations of BSA (0.05–0.1 �g/�l)could eliminate inhibition in strawberry extractions, but notin the blackberry extractions. Larger concentrations of BSA(3–10 �g/�l) removed inhibition in both blackberry and strawberrybut suppressed PCR amplification, especially at smaller cDNA con-centrations (data not shown). A final concentration of BSA between0.5 and 1 �g/�l was found to be optimal for eliminating inhibitorsand enhancing the efficiency of PCR (data not shown), includingtargets (RpRSV and TBSV) whose primers lacked any potential toproduce secondary structures (Table 2).
3.3. Primer interaction and mitigation of interaction in multiplexPCR
3.3.1. Impact of primer interaction on PCR efficiency using singleand multiple primer pairs at different concentrations withindividual cDNA templates
To investigate the impact of primer interactions on PCR effi-ciency in multiplex PCR, simplex and multiplex PCR on single cDNAtemplates were performed using different primer concentrations attwo different extension temperatures (65 and 72 ◦C) and an anneal-
ing temperature of 56 ◦C. Results showed that primer interactionsexist in multiplex PCR, especially at larger primer concentrationsand higher extension temperatures (72 ◦C), which resulted in areduced amplification efficiency (Fig. 1). Primer concentrationsbetween 125 and 1000 nM/primer in simplex PCR gave a similaryield of amplicons at both extension temperatures but a slightlysmaller amount when combined with PIAC (Fig. 1). For PIAC, theamplification efficiency was generally suppressed when combinedwith each of the three viruses. When primer concentrations werereduced, amplification efficiency of PIAC was decreased in combi-nation with RpRSV and SLRSV but increased in combination withTBSV (Fig. 1). Conversely, increasing the primer concentration insimplex PCR increased PCR efficiency when cDNA concentrationswere small (data not shown). Primer dimers were not observedgenerally except when using the largest primer concentrations ofSLRSV and TBSV in multiplex PCR (Fig. 1).
The impact of primer interaction on PCR efficiency was inves-tigated further using mixed primers for the three viruses atdifferent concentrations with individual cDNA templates. Theresults showed that primer interactions were stronger at largerprimer concentrations and thus amplification of all targets
T. Wei et al. / Journal of Virological Methods 151 (2008) 132–139 135
n combination with PIAC at extension temperatures of 65 and 72 ◦C. Lanes 1–5: Primerprimer concentrations of virus and PIAC primers at 500, 250, 125, and 62.5 nM/primer,
Fig. 1. Primer interaction and PCR amplification efficiency for individual viruses iconcentration at 1000, 500, 250, 125 and 62.5 nM/primer, respectively; lanes 6–9:respectively. M: 100 bp DNA ladder (Invitrogen).
increased as primer concentrations decreased (in a range of125–1660 nM/primer), with the best amplifications of all targetsbeing achieved at concentrations of 125–250 nM/primer (Fig. 2).More primer dimers were observed at larger primer concentra-tions, in particular with the SLRSV primers which had an annealingenergy with all other primers.
3.3.2. Impact of primer interaction on PCR efficiency usingmultiple primer pairs at different concentrations with multiplecDNA templates
Primer interaction was investigated subsequently using two,three and four pairs of primers at different concentrations on rele-vant multiple targets. Primers concentrations ranged from 62.5 to250 nM/primer based on the results reported in Section 3.3.1. Aspreviously observed, primer interactions were stronger at largerprimer concentrations and better amplification of multiple targetswas always achieved at smaller primer concentrations, either 62.5or 125 nM/primer (lanes 2, 3, 9, 11, 12, 14, and 15 in Fig. 3A). Veryfaint primer dimers were observed only when three or four pairs
Fig. 2. Primer interaction and PCR amplification efficiency for individual virusesusing three primer pairs at different concentrations. Lanes 1–3, 4–6, 7–9, 10–12,13–15, 16–18 and 19–21 represent primer concentrations of 1660, 830, 415, 250,125, 83 and 41.5 nM/primer, respectively; lane M: 100 bp DNA ladder (Invitrogen).
Table 3The annealing free energy (�G: kcal/mol) and homology (%) between different primers p
of primers were present at the largest primer concentration (lanes10 and 13 in Fig. 3A).
Preferential amplification of one target over another occurredwith all pairs of primers and at all primer concentrations, regard-less of the predicted secondary structures. For example, RpRSV was
Fig. 3. Primer interaction and PCR amplification efficiency for multiple targets usingdifferent primer pairs at different concentrations. Panel A: Primer interaction andPCR amplification efficiency for two, three and four targets at different primer con-centrations. Lanes 1, 4, 7, 10: 250 nM/primer; lanes 2, 5, 8, 11 and 14: 125 nM/primer;lanes 3, 6, 9, 12 and 15: 62.5 nM/primer. Panel B: Efficiency of PCR amplification effi-ciency using different primer combinations on four targets. Primer concentrationsfor RpRSV, SLRSV, TBSV and PIAC are 100, 250, 125 and 62.5 nM, respectively. Lanes1–3 represent three mixed cDNA templates at similar concentrations, 1:10 dilutionof mixed cDNA and 1:100 dilution of mixed cDNA, respectively.
four targets amplified by the relevant primer pairs. Lanes 1–5 represent differentextension temperatures of 60, 63, 66.7, 69 and 71.7 ◦C, respectively. Panel B: Differ-ent extension periods at an extension temperature of 65 ◦C. Lanes 1 and 2, 3 and 4,and 5 and 6 represent two different dilutions (1: 20,000 and 1:40,000) for RpRSV,SLRSV and TBSV, respectively.
1:3 diluted cDNA and 1:1.5 diluted cDNA for RpRSV, SLRSV andTBSV, respectively). These detection limits are comparable to thosefor individual viruses using the published protocols (1:10,000,1:200,000, 1:80,000 for RpRSV, SLRSV and TBSV, respectively).
3.4. Application of the multiplex PCR in simulated field conditions
The multiplex PCR was evaluated further using two or threecDNA templates at different concentrations to simulate field infec-tions. In samples containing two templates, the detection limits
136 T. Wei et al. / Journal of Virolo
Fig. 4. Simplex and multiplex PCR efficiencies with the addition of BSA. Efficien-cies of simplex and multiplex PCRs for all targets were increased by the addition of0.5 �g/�l. Lanes 1–3: four pairs of mixed primers with individual cDNA templates ofRpRSV, SLRSV and TBSV, respectively; lanes 4–6: Four pairs of mixed primers withmixed cDNA templates of RpRSV and SLRSV, RpRSV and TBSV, and SLRSV and TBSV,respectively; lane 7: Four pairs of mixed primers with mixed cDNA templates ofRpRSV, SLRSV and TBSV.
amplified preferentially compared to SLRSV (lanes 1–3 in Fig. 3A),and TBSV was amplified preferentially compared to RpRSV (lanes4–6 in Fig. 3A) and SLRSV (lanes 7–8 in Fig. 3A). When the mixedtemplates were diluted further, a smaller primer concentration(62.5 nM/primer) showed more efficient amplification to all targetsthan a larger concentration (125 nM/primer) (Fig. 3B).
Different combinations of primer concentrations were alsoinvestigated by reducing the concentrations of the primer pairswhich provided preferential amplification, i.e. those for RpRSV,TBSV and PIAC (Fig. 3). The results showed that primer pair con-centrations of 100, 250, 125 and 80 nM for RpRSV, SLRSV, TBSVand PIAC, respectively provided the best amplification efficiency forfour targets, especially at smaller template concentrations (Fig. 3B).Other combinations in which all primers were present at eitherlarger (500 nM/primer) or smaller (125 nM/primer) concentrationsdid not detect all targets especially at smaller template concentra-tions (data not shown).
3.3.3. Impact of primer interaction on PCR efficiency using BSAusing different PCR conditions
As reported in Section 3.2, addition of BSA can improve thePCR efficiency for individual viruses. The effect of BSA on primerinteraction was investigated by adding 0.5 �g/�l BSA (final con-centration) to simplex and multiplex PCR. The results showed thatthe amplification efficiency of all targets was increased (Fig. 4).Stronger primer interactions were observed at higher extension
temperatures which resulted in a smaller amplification efficiency(Fig. 5A). Extension temperatures ranging from 60 to 66.7, 60 to 69,and 63 to 66.7 ◦C provided better amplification of individual viruses(Fig. 5A1), individual viruses and PIAC (Fig. 5A2), and all of the fourtargets (Fig. 5A3), respectively. The amplification efficiency of eithersingle or multiple targets within the investigated extension tem-perature range was not affected when cDNA concentrations wereincreased (data not shown). The amplification efficiency of indi-vidual or multiple targets was greater if the extension period wasincreased (Fig. 5B) but was unaffected by increasing the annealingperiod (data not shown). Increasing the number of PCR cycles to 45increased the amplification of each target (data not shown).
The optimal PCR conditions were: (1) primer concentrations forRpRSV, SLRSV, TBSV and PIAC at 100, 250, 125 and 80 nM, respec-tively; (2) 0.5 �g/�l BSA in PCR mixture; and (3) thermo-cyclingat 94 ◦C for 3 min, followed by 45 cycles of denaturing at 94 ◦C for20 s, annealing at 56 ◦C for 20 s, and extension at 65 ◦C for 3 min,plus a final extension step at 72 ◦C for 5 min. The detection limitof the multiplex PCR was 1:64,000, 1:192,000 and 1:48,000 forRpRSV, SLRSV and TBSV, respectively (Fig. 6) when cDNAs werepresent at similar concentrations (equating to undiluted cDNA,
Fig. 5. Primer interaction and PCR amplification efficiency at different extensionconditions. Greater amplification efficiency for single and multiple targets wasobtained at smaller extension temperatures ranging from 63 to 66.7 ◦C (A) andlonger extension periods (B). Panel A: Different extension temperatures—A1: indi-vidual virus target amplified by single primer pairs; A2: two targets (individualvirus + PIAC) amplified by the relevant primer pairs; A3: three virus targets or all
for templates at the smaller concentration were generally less andranged from 1:400 to 1:1000 (Table 4). In samples containing twocDNA templates in large concentration and the third at a smallerconcentration, the detection limits for the targets at the smallerconcentration were 1:200, 1:800, and 1:400 for RpRSV, SLRSV, andTBSV, respectively (Table 4). Similarly, the detection limits were
Fig. 6. Detection limits of developed multiplex PCR with different PCR cycling condi-tions. Three cDNAs were mixed in similar quantities. The detection limits for RpRSV,SLRSV and TBSV were 1:16,000, 1:48,000 and 1:24,000, respectively with 40 PCRcycles and 1:32,000, 1:96,000 and 1:48,000, respectively with 45 PCR cycles. Lane1: Mixed cDNA of RpRSV (original) + SLRSV (1:3) + TBSV (1:1.5); lanes 2–10: 1:10,1:100, 1:1000, 1:2000, 1:4000, 1:8000, 1:16,000, 1:32,000 and 1:64,000 dilutionsof mixed cDNA templates, respectively.
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Table 4Detection limits of multiplex PCR on individual and multiple cDNA templates at dif
Three cDNA mixture (low + low + low) Low (25,600–512,00
1:400, 1:8000–1:10,000, and 1:400 for RpRSV, SLRSV, and TBSV,respectively when two templates were at a smaller concentrationand the third at a large concentration. These results show that thedetection limit for multiple templates cannot be compared withthe detection limit for the single virus when the cDNA templates arepresent in different concentrations. However, this problem could beovercome by diluting the primer concentrations for those viruseswhich were amplified preferentially (data not shown), confirmingthat primer interaction is the crucial factor affecting amplificationefficiency in multiplex PCR. When the templates were present insimilar concentrations, the detection limits for all the targets werecomparable to those of individual viruses.
4. Discussion
Various approaches have been used to overcome the difficul-ties encountered in the development of multiplex PCR (Brownie etal., 1997; Zheng et al., 1995). These approaches have been basedmainly on optimisation of primer design (Chavali et al., 2005; Maand Michailides, 2007), elimination of primer dimer formation(Chou et al., 1992; Shigemori et al., 2005), and removal of PCRinhibitors (Eckhart et al., 2000; Forbes and Hicks, 1996; Singh etal., 2002). This study has developed a strategy to address theseproblems by identifying the key factors affecting amplification effi-ciency in multiplex PCR. The strategy has been used to develop asensitive multiplex PCR for the simultaneous detection of RpRSV,
SLRSV and TBSV in strawberry, including an internal amplificationcontrol.
PCR inhibitors are composed of various substances and existwidely in a variety of samples such as humic acid from soil (Chavaliet al., 2005; Desai and Madamwar, 2007), EDTA, heme compoundsand sodium polyanetholesulfonate from blood or blood culturemedia (Akane et al., 1994; Fredricks and Relman, 1998), urine (Khanet al., 1991), and polysaccharides and polyphenolics from plant tis-sues (Demeke and Adams, 1992; Malvick and Grunden, 2005). Themechanism of inhibition is considered to be chelation of the Mg2+
cofactor which is important for Taq polymerase activity, or by bind-ing to target DNA or polymerase (Wilson, 1997). Alternatively, plantsecondary metabolites may precipitate Taq polymerase resulting inreduced activity. Plant polyphenolics also inhibit Taq polymeraseby forming secondary structures through the phenol rings (Mayret al., 2005) which results in the inactivation of the enzyme andPCR failure. Attempts have been taken to eliminate PCR inhibitionby modifying RNA extraction methods (John, 1992; Koonjul et al.,1999; Singh et al., 2002; Thompson et al., 2003). However, theseapproaches are often time-consuming and can cause loss of targetnucleic acids. Immunocapture PCR may be used to avoid the impact
ethods 151 (2008) 132–139 137
concentrations
SLRSV (S) TBSV (T)
At least 1:102,400 At least 1:51,200Low (1:1000) –– Low (1:1000)High –High Low (1:400)– HighLow (1:400) High
High Low (1:200)Low (1:800) HighHigh High
Low (1:8000) Low (1:400)High Low (1:400)Low (1:10,000) High
Low (∼100,000) Low (48,000–72,000)
of inhibitors (Vigano and Stevens, 2007) but requires specific anti-sera.
In this study, inhibition of the PCR by plant extract(s) from straw-berry and in particular, blackberry was observed. A 1:100 dilutionfor blackberry cDNA and 1:10 dilution for strawberry cDNA wasrequired for the successful amplification of the target fragments(data not shown). It was found that addition of 0.5–1 �g/�l BSA tothe PCR mixture removed inhibition effectively and improved theamplification efficiency. BSA has been reported to eliminate PCRinhibition in other tissues (Desai and Madamwar, 2007; Eckhartet al., 2000; Forbes and Hicks, 1996) but rarely in plant samples.The mechanism postulated previously was that BSA prevented sur-face denaturation of the polymerase (Malhotra et al., 1998). In thisstudy, it is speculated that the amino residues of lysines on BSA(Huang et al., 2004) bind to the carbonyl groups of polyphenolicsby Schiff base formation thereby preventing the inactivation of TaqDNA polymerase. However, excess BSA was found to inhibit PCRamplification. The mechanism for this is unclear but may be due toBSA blocking Taq polymerase’s access to the primer-binding sites.
A successful multiplex PCR requires all primers to have simi-lar optimal annealing temperatures and no significant homologyeither internally or to each other (Ma and Michailides, 2007). Inthis study, computer analysis suggested that self-annealing, self-looping and annealing between forward and reverse primer pairsrelated to the formation of putative secondary structure would beunlikely to affect amplification in simplex PCR. This is supported by
the reports that the four primer pairs were able to amplify their tar-get fragments with high specificity (Harris et al., 2006; Menzel et al.,2002; Ochoa-Corona et al., 2006; Postman et al., 2004). In addition,the analysis indicated that Tms of all primers for viral targets weresimilar enough (58–62 ◦C) to allow the same annealing tempera-ture. This was demonstrated in simplex PCR in which all primersamplified their respective templates with the best efficiency at anannealing temperature of 56 ◦C. As expected, the limited homologybetween primer pairs (less than 50%) did not cause non-specificamplification (Kane et al., 2000).
Primer interaction is a crucial factor affecting the successfuldevelopment of multiplex PCR. Although many computer pro-grammes have been developed to predict potential secondarystructures (Brownie et al., 1997; Chou et al., 1992; Shigemori et al.,2005), in practice it is still difficult to choose primers without suchpotential problems. In this study, we have demonstrated empiri-cally that primer interaction is a key factor in: (1) reducing simplexPCR efficiency using the RpRSV and TBSV primers which had nopredicted secondary structures (Fig. 4); (2) simplex and multiplexPCR efficiencies at larger extension temperatures (Figs. 1 and 5A);and (3) multiplex PCR efficiency at large primer concentrations
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138 T. Wei et al. / Journal of Virolo
(Figs. 1–3A). Primer secondary structures including primer dimers,self-annealing and self-looping are known to cause this reduction inPCR amplification efficiency. This phenomenon was observed whenusing the SLRSV primers, and between other primer pairs in multi-plex PCR, especially at larger primer concentrations (Figs. 1 and 3A).
At greater primer concentrations (Figs. 1–3A), larger extensiontemperatures (Figs. 1 and 5A), or without the addition of BSA(Fig. 4), amplification efficiency was reduced, even when usingprimers with no self-annealing, self-looping or annealing betweendifferent primers (e.g. the RpRSV and TBSV primers). However, inthis situation, no or very little primer dimer was observed. Theseresults indicate that there is another kind of invisible primer inter-action which can reduce PCR amplification efficiency but whichhas not been reported previously. This primer interaction was des-ignated “primer cluster”. The results indicate that such “primerclusters” are the main factor reducing amplification efficiency. Theeffect of primer dimers and “primer clusters” could be mitigatedby reducing primer concentration especially those which preferen-tially amplify (Figs. 1–3), decreasing the PCR extension temperature(Figs. 1 and 5A), increasing the PCR extension period (Fig. 5B) andadding an optimal concentration of BSA to the PCR mixture (Fig. 4).These approaches increased significantly the amplification effi-ciency of all target fragments in both simplex and multiplex PCRfor the detection of individual or multiple targets.
The concept of decreasing the primer concentration to reducethe interaction between primers was drawn from the principleof PCR amplification. In the PCR process, the interaction betweenprimers and/or templates to form primer–primer secondarystructures or primer–template duplexes results from molecular col-lision. At the same temperature, small molecules (primers) canmove faster than large molecules (templates). Therefore at largerprimer concentrations, there are more opportunities for the fastermoving primers to interact with each other to form primer dimersor “primer clusters” rather than hybridising with templates; thisresults in reduced amplification efficiency. A decrease in primerconcentration should reduce the opportunity for primers to inter-act with each other and thus increase the opportunities for primersto interact with their templates resulting in an increased amplifica-tion efficiency, especially when combined with reduced extensiontemperatures, longer extension periods and increased PCR cycles.This is particularly the case at larger extension temperaturesdue to the accelerated molecular movement resulting in unstableprimer–template duplexes and the release of more primers to formprimer dimers or “primer clusters”. Longer extension periods of up
to 3 min but not longer annealing periods were found to increasePCR efficiency of both single and multiple targets in multiplex PCR.These results suggest that a longer extension period might increasethe amount of stable primer–template duplexes for amplification.
The addition of BSA in PCR was found to improve amplificationefficiency (Fig. 4). This may be due to the effect of BSA absorbingplant inhibitors or reducing the adsorption of Taq polymerase tothe PCR tube surface (Erill et al., 2003). However with the sameamount of Taq polymerase, amplification efficiency depends onprimer concentration when there are several templates (Fig. 1). It ispostulated that BSA may change the PCR buffer stringency resultingin the degeneration of primer secondary structure and the releaseof more primers which can amplify more targets. This is supportedby previous research which shows that a change in buffer strin-gency can result in the unstable binding of DNA-duplex (Liu et al.,2001; Relogio et al., 2002).
Although sensitivity and specificity are critical parameters inmultiplex PCR, little research has been done on these aspects. Basedon our research, some published multiplex PCRs can work onlywhen all templates are present in large concentration. In this study,we predicted in silico the potential to develop a multiplex PCR
ethods 151 (2008) 132–139
by analysing the characteristics of published primers. The resultsshowed that it was possible to develop a multiplex PCR for the fourtargets with high specificity but there might be less sensitivity fortargets whose primers had greater self-annealing, self-looping andannealing with other primers. Initial experiments in which an equalconcentration of each primer was used confirmed these predic-tions (data not shown). However, the sensitivity of multiplex PCRfor each target was increased dramatically using the approachesdescribed above and resulted in sensitivity comparable to simplexPCR. The detection limit(s) for template(s) in small concentrationwas decreased when mixed with one or more template(s) at largerconcentration(s). However, this decrease in detection limit may notaffect its application in quarantine situations where the presenceof any of three viruses may be unacceptable. The decrease in sen-sitivity could be reduced to reach similar limits to simplex PCRby diluting the primers for those viruses present in greater con-centrations (preferential amplification targets). However, there aredifficulties in predicting the relative concentrations of each virusin field samples and therefore the applicability of this method indiagnostic testing should be verified prior to routine adoption.
In conclusion, this study developed approaches to mitigateprimer interaction and eliminate PCR inhibitors, including reduc-ing primer concentration (especially those with preferentialamplification) and extension temperature, the use of BSA, in com-bination with increasing the extension time and PCR cycles. Theseapproaches have been used to develop a multiplex PCR methodfor the detection of RpRSV, SLRSV and TBSV in strawberry with aninternal amplification control, and with the same specificity andcomparable sensitivity as simplex PCR. These approaches can beapplied to the development of sensitive multiplex PCR assays forpathogens from plants, animals and the environment. They alsoprovide a simple way for the development of multiplex PCR byenabling the use of published primers rather than designing novelprimers. The results confirmed that analysis of the primer charac-teristics is useful in predicting the potential to develop multiplexPCR.
Acknowledgements
We thank Dr. Zhidong Yu in the Growth and Development sec-tion, AgResearch, New Zealand; Dr. Lei Zhang in the Dental ResearchInstitute, University of California, Los Angeles; and Dr. FranciscoOchoa-Corona and Dr. Mark Braithwaite in MAF Biosecurity New
Zealand for revising the manuscript. We also thank our colleaguesDr. Francisco Ochoa-Corona, Mr. Joe Tang and Dr. Lisa Ward forproviding information on the samples and primers.
References
Akane, A., Matsubara, K., Nakamura, H., Takahashi, S., Kimura, K., 1994. Identifica-tion of the heme compound copurified with deoxyribonucleic acid (DNA) frombloodstains, a major inhibitor of polymerase chain reaction (PCR) amplification.J. Forensic Sci. 39, 362–372.
Barritt, B.H., Loo, H.Y.S., 1973. Effects of mottle, crinkle, and mild yellow-edge viruseson growth and yield of Hood and Northwest strawberries. Can. J. Plant Sci. 53,605–607.
Brownie, J., Shawcross, S., Theaker, J., Whitcombe, D., Ferrie, R., Newton, C., Little, S.,1997. The elimination of primer–dimer accumulation in PCR. Nucleic Acids Res.25, 3235–3241.
Cadman, C.H., 1956. Studies on the etiology and mode of spread of Scottish raspberryleaf curl disease. J. Hort. Sci. 31, 111–118.
Chavali, S., Mahajan, A., Tabassum, R., Maiti, S., Bharadwaj, D., 2005. Oligonucleotideproperties determination and primer designing: a critical examination of pre-dictions. Bioinformatics 21, 3918–3925.
Chou, Q., Russel, M., Birch, D.E., Raymond, B.J., Bloch, W., 1992. Prevention of pre-PCRmis-priming and primer dimerization improves low-copy-number amplifica-tions. Nucleic Acids Res. 11, 1717–1723.
Demeke, T., Adams, R.P., 1992. The effects of plant polysaccharides and buffer addi-tives on PCR. BioTechniques 12, 332–334.
gical M
T. Wei et al. / Journal of Virolo
Desai, C., Madamwar, D., 2007. Extraction of inhibitor-free metagenomic DNA frompolluted sediments, compatible with molecular diversity analysis using adsorp-tion and ion-exchange treatments. Bioresour. Technol. 98, 761–768.
Eckhart, L., Bach, J., Ban, J.F., Tschachler, E., 2000. Melanin binds reversibly tothermostable DNA polymerase and inhibits its activity. Biochem. Biophys. Res.Commun. 271, 726–730.
Erill, I., Campoy, S., Erill, N., Barbe, J., Aguilo, J., 2003. Biochemical analysis and opti-mization of inhibition and adsorption phenomena in glass-silicon PCR-chips.Sens. Actuators B: Chem. 96, 685–692.
Forbes, B.A., Hicks, K.E., 1996. Substances interfering with direct detection ofMycobacterium tuberculosis in clinical specimens by PCR: effects of bovine serumalbumin. J. Clin. Microbiol. 34, 2125–2128.
Fredricks, D.N., Relman, D.A., 1998. Improved amplification of microbial DNA fromblood cultures by removal of the PCR inhibitor sodium polyanetholesulfonate. J.Clin. Microbiol. 36, 2810–2816.
Harris, R., Ochoa-Corona, R., Lebas, B., Timudo, O., Stewart, F., Alexander, B., 2006.Broad detection and diagnosis of viruses of the genus Tombusvirus by RT-PCRcoupled to single strand conformation polymorphism analysis. In: Proceedingsof the 7th Australasian Plant Virology Workshop, Rottnest Island, Perth, Aus-tralia, November 8–11, 2006, p. 48.
Huang, B.X., Kim, H.Y., Dass, C., 2004. Probing three-dimensional structure of bovineserum albumin by chemical cross-linking and mass spectrometry. J. Am. Soc.Mass Spectrom. 15, 1237–1247.
John, M.E., 1992. An efficient method for isolation of RNA and DNA from plant con-taining polyphenolics. Nucleic Acids Res. 20, 2381.
Kaitasowa, P., Kondakowa, V., 1989. Immune electron microscopic detection oftomato bushy stunt virus in strawberries. Arch. Phytopathol. Pflanzenschutz 25,515–516.
Kane, M.D., Jatkoe, T.A., Stumpf, C.R., Lu, J., Thomas, J.D., Madore, S.J., 2000. Assess-ment of the sensitivity and specificity of oligonucleotide (50mer) microarrays.
Nucleic Acids Res. 28, 4552–4557.
Khan, G., Kangro, H.O., Coates, P.J., Heath, R.B., 1991. Inhibitory effects of urine on thepolymerase chain reaction for cytomegalovirus DNA. J. Clin. Pathol. 44, 360–365.
Koonjul, P.K., Brandt, W.B., Farrant, J.M., Lindsey, G.G., 1999. Inclusion of polyvinylpyrrolidone in the polymerase chain reverses the inhibitory effects of polyphe-nolic contamination of RNA. Nucleic Acids Res. 27, 915–916.
Li, X., Huang, Y., Song, C., Zhao, M., Li, Y., 2007. Several concerns about the primerdesign in the universal molecular beacon real-time PCR assay and its applicationin HBV DNA detection. Anal. Bioanal. Chem. 388, 979–985.
Lister, R.M., 1964. Strawberry latent ringspot: a new nematode-borne virus. Ann.Appl. Biol. 54, 167–176.
Liu, W.T., Mirzabekov, A.D., Stahl, D.A., 2001. Optimization of an oligonucleotidemicrochip for microbial identification studies: a non-equilibrium dissociationapproach. Environ. Microbiol. 3, 619–629.
Ma, Z., Michailides, T.J., 2007. Approaches for eliminating PCR inhibitors and design-ing PCR primers for the detection of phytopathogenic fungi. Crop Prot. 26,145–161.
Malhotra, K., Foltz, L., Mahoney, W.C., Shueler, P.A., 1998. Interaction and effect ofannealing temperature on primers used in differential display RT-PCR. NucleicAcids Res. 26, 854–856.
Malvick, D.K., Grunden, E., 2005. Isolation of fungal DNA from plant tissues andremoval of DNA amplification inhibitors. Mol. Ecol. Notes 5, 958–960.
Martin, R.R., Tzanetakis, I.E., 2006. Characterization and recent advances in detectionof strawberry viruses. Plant Dis. 90, 384–396.
Mayr, G.W., Winidhorst, S., Hillemeier, K., 2005. Antiproliferative plant andsynthetic polyphenolics are specific inhibitors of vertebrate inositol-1,4,5-trisphosphate 3-kinases and inositol polyphosphate multikinase. J. Biol. Chem.280, 13229–13240.
ethods 151 (2008) 132–139 139
Menzel, W., Jelkmann, W., Maiss, E., 2002. Detection of four apple viruses by mul-tiplex RT-PCR assays with coamplification of plant mRNA as internal control. J.Virol. Methods 99, 81–92.
Narayanasamy, P., 1997. Plant Pathogen Detection and Disease Diagnosis. MarcelDekker, Inc., New York.
Ochoa-Corona, F.M., Lebas, B.S.M., Tang, J.Z. Bootten, T.J., Stewart, F.J., Harris, R.,Elliott, D.R., Alexander, B.J.R., 2006. RT-PCR detection and strain typing ofRaspberry ringspot virus. Proceedings of the XXth International Symposiumon Virus and Virus-like Diseases of Temperate Fruit Crops & XIth Interna-tional Symposium on Small Fruit Virus Diseases, Antalya, Turkey, May 22–26,2006.
Polz, M.F., Cavanaugh, C.M., 1998. Bias in template-to-product ratios in multitem-plate PCR. Appl. Environ. Microbiol. 64, 3724–3730.
Porebski, S., Bailey, L.G., Baum, B.R., 1997. Modification of a CTAB DNA extractionprotocol for plants containing high polysaccharide and polyphenol components.Plant Mol. Biol. Rep. 15, 8–15.
Postman, J.D., Tzanetakis, I.E., Martin, R.R., 2004. First report of Strawberry latentringspot virus in a Mentha sp. from North America. Plant Dis. 88, 907.
Puskas, L.G., Bottka, S., 1995. Reduction of mispriming in amplification reactionswith restricted PCR. Genome Res. 5, 309–311.
Relogio, A., Schwager, C., Richter, A., Ansorge, W., Valcarcel, J., 2002. Optimization ofoligonucleotide-based DNA microarrays. Nucleic Acids Res. 30, e51.
Roy, A., Fayad, A., Barthe, G., Brlansky, R.H., 2005. A multiplex polymerase chainreaction method for reliable, sensitive and simultaneous detection of multipleviruses in citrus trees. J. Virol. Methods 129, 47–55.
Rychlik, W., 1995. Selection of primers for polymerase chain reaction. Mol. Biotech-nol. 3, 129–134.
Sanchez-Navarro, J.A., Aparicio, F., Herranz, M.C., Minafra, A., Myrta, A., Pallas, V.,2005. Simultaneous detection and identification of eight stone fruit viruses byone-step RT-PCR. Eur. J. Plant Pathol. 111, 77–84.
Seeram, N.P., Adams, L.S., Zhang, Y., Lee, R., Sand, D., Scheuller, H.S., Heber, D., 2006.Blackberry, black raspberry, blueberry, cranberry, red raspberry, and strawberryextracts inhibit growth and stimulate apoptosis of human cancer cells in vitro.J. Agric. Food Chem. 54, 9329–9339.
Shigemori, Y., Mikawa, T., Shibata, T., Oishi, M., 2005. Multiplex PCR: use ofheat-stable Thermus thermophilus RecA protein to minimize non-specific PCRproducts. Nucleic Acids Res. 33, e126.
Singh, R.P., Nie, X., Singh, M., Coffin, R., Duplessis, P., 2002. Sodium sulphite inhibitionof potato and cherry polyphenolics in nucleic acid extraction for virus detectionby RT-PCR. J. Virol. Methods 99, 123–131.
Stevens, M., Hull, R., Smith, H.G., 1997. Comparison of ELISA and RT-PCR for thedetection of beet yellow closterovirus in plants and aphids. J. Virol. Methods 68,9–16.
Thomson, D., Dietzgen, R.G., 1995. Detection of DNA and RNA plant viruses by PCRand RT-PCR using a rapid virus release protocol without tissue homogenization.J. Virol. Methods 54, 85–95.
Thompson, J.R., Wetzel, S., Klerks, M.M., Vaskova, D., Schoen, C.D., Spak, J., Jelkmann,W., 2003. Multiplex RT-PCR detection of four aphid-borne strawberry viruses inFragaria spp. in combination with a plant mRNA specific internal control. J. Virol.Methods 111, 85–93.
Vigano, F., Stevens, M., 2007. Development of a multiplex immunocapture-RT-PCRfor simultaneous detection of BMYV and BChV in plants and single aphids. J.Virol. Methods 146, 196–201.
Wagner, A., Blackstone, N., Cartwright, P., Dick, M., Misof, B., Snow, P., Wagner, G.P.,Bartels, J., Murtha, M., Pendleton, J., 1994. Surveys of gene families using poly-merase chain reaction: PCR selection and PCR drift. Syst. Biol. 43, 250–261.
Wilson, I.G., 1997. Inhibition and facilitation of nucleic acid amplification. Appl.Environ. Microbiol. 63, 3741–3751.
Zheng, P.S., Li, S.R., Iwasaka, T., Song, J., Gui, M.H., Sugimori, H., 1995. Simultaneousdetection by consensus multiplex PCR of high- and low-risk and other types ofhuman papilloma virus in clinical samples. Gynecol. Oncol. 58, 179–183.
