Abstract
SYBR Green I (SG) is widely used in real-time PCR applications as an intercalating dye. Preferential binding of SG during PCR and inhibition of PCR often result in failure to detect multiple amplicons in multiplex reactions. In the present study, a novel single-tube, multiplex real-time PCR with EvaGreen dye (EG) was developed and evaluated for simultaneous detection of pathogenic targets by using five potato viruses as models. The PCR products obtained using five sets of specific primers were analyzed by melting curve analysis. The assay could specifically detect and differentiate the five potato viruses by producing a distinct peak for each amplification product and exhibited a high reproducibility with coefficients of variation from 0.01 to 0.25 %. Detection sensitivity of the assay ranged from 100 to 500 copies/μL for each virus. The results of this study demonstrate that multiplex real-time PCR and melting-curve analysis with EG is a sensitive, specific and inexpensive method for simultaneous detection of multiple pathogens.
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References
Agindotan BO, Shiel PJ, Berger PH (2007) Simultaneous detection of potato viruses, PLRV, PVA, PVX and PVY from dormant potato tubers by TaqMan real-time RT-PCR. J Virol Methods 142:1–9
Armani A, Castigliego L, Tinacci L, Gianfaldoni D, Guidi A (2012) Multiplex conventional and real-time PCR for fish species identification of Bianchetto (juvenile form of Sardina pilchardus), Rossetto (Aphia minuta), and Icefish in fresh, marinated and cooked products. Food Chem 133:184–192
Boonham N, Walsh K, Mumford RA, Barker I (2000) Use of multiplex real-time PCR (TaqMan) for the detection of potato viruses. EPPO Bull 30:427–430
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
Bustin SA (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25:169–193
Bustin SA (2002) Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J Mol Endocrinol 29:23–39
Chassagne L, Pradel N, Robin F, Livrelli V, Bonnet R, Delmas J (2009) Detection of stx1, stx2, and eae genes of enterohemorrhagic Escherichia coli using SYBR Green in a real-time polymerase chain reaction. Diagn Microbiol Infect Dis 64:98–101
Du ZY, Chen JS, Hiruki C (2006) Optimization and application of a multiplex RT-PCR System for simultaneous detection of five potato viruses using 18S rRNA as an internal control. Plant Dis 90:185–189
Elizaquivel P, Aznar R (2008) A multiplex RTi-PCR reaction for simultaneous detection of Escherichia coli O157:H7, Salmonella spp. and Staphylococcus aureus on fresh, minimally processed vegetables. Food Microbiol 25:705–713
Giglio S, Monis PT, Saint CP (2003) Demonstration of preferential binding of SYBR Green I to specific DNA fragments in real-time multiplex PCR. Nucleic Acids Res 31:e136
Guion CE, Ochoa TJ, Walker CM, Barletta F, Cleary TG (2008) Detection of diarrheagenic Escherichia coli by use of melting-curve analysis and real-time multiplex PCR. J Clin Microbiol 46:1752–1757
Heid CA, Stevens J, Livak KJ, Williams PM (1996) Real time quantitative PCR. Genome Res 6:986–994
Socorro Hernandez-Arteaga, Rubén L-R (2008) Quantitation of human papilloma virus type 16 E6 oncogene sequences by real-time or quantitative PCR with EvaGreen. Anal Biochem 380:131–133
Higuchi R, Dollinger G, Walsh PS, Griffith R (1992) Simultaneous amplification and detection of specific DNA sequences. Bio/Technology 10:413–417
Khan SA, Sung K, Nawaz MS (2011) Detection of aacA-aphD, qacEdelta1, marA, floR, and tetA genes from multidrug-resistant bacteria: Comparative analysis of real-time multiplex PCR assays using EvaGreen® and SYBR® Green I dyes. Mol Cell Probes 25:78–86
Li YD, Chu ZZ, Liu XG, Jing HC, Liu YG, Hao DY (2010) A cost-effective high-resolution melting approach using the EvaGreen dye for DNA polymorphism detection and genotyping in plants. J Integr Plant Biol 52:1036–1042
Lipsky RH, Mazzanti CM, Rudolph JG, Xu K, Vyas G, Bozak D, Radel MQ, Goldman D (2001) DNA melting analysis for detection of single nucleotide polymorphisms. Clin Chem 47:635–644
Mao F, Leung WY, Xin X (2007) Characterization of EvaGreen and the implication of its physicochemical properties for qPCR applications. BMC Biotechnol 7:76
Mendes RE, Kiyota KA, Monteiro J, Castanheira M, Andrade SS, Gales AC, Pignatari AC, Tufik S (2007) Rapid detection and identification of metallo-beta-lactamase-encoding genes by multiplex real-time PCR assay and melt curve analysis. J Clin Microbiol 45:544–547
Monis PT, Giglio S, Saint CP (2005) Comparison of SYTO9 and SYBR Green I for real-time polymerase chain reaction and investigation of the effect of dye concentration on amplification and DNA melting curve analysis. Anal Biochem 340:24–34
Mortimer-Jones SM, Jones MG, Jones RA, Thomson G, Dwyer GI (2009) A single tube, quantitative real-time RT-PCR assay that detects four potato viruses simultaneously. J Virol Methods 161:289–296
Papin JF, Vahrson W, Dittmer DP (2004) SYBR green-based real-time quantitative PCR assay for detection of West Nile Virus circumvents false-negative results due to strain variability. J Clin Microbiol 42:1511–1518
Perez LJ, Perera CL, Frias MT, Nunez JI, Ganges L, de Arce HD (2012) A multiple SYBR Green I-based real-time PCR system for the simultaneous detection of porcine circovirus type 2, porcine parvovirus, pseudorabies virus and Torque teno sus virus 1 and 2 in pigs. J Virol Methods 179:233–241
Pietila J, He Q, Oksi J, Viljanen MK (2000) Rapid differentiation of Borrelia garinii from Borrelia afzelii and Borrelia burgdorferi sensu stricto by LightCycler fluorescence melting curve analysis of a PCR product of the recA gene. J Clin Microbiol 38:2756–2759
Polz MF, Cavanaugh CM (1998) Bias in template-to-product ratios in multitemplate PCR. Appl Environ Microbiol 64:3724–3730
Ririe KM, Rasmussen RP, Wittwer CT (1997) Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem 245:154–160
Tanriverdi S, Tanyeli A, Baslamisli F, Koksal F, Kilinc Y, Feng X, Batzer G, Tzipori S, Widmer G (2002) Detection and genotyping of oocysts of Cryptosporidium parvum by real-time PCR and melting curve analysis. J Clin Microbiol 40:3237–3244
Vandesompele J, De Paepe A, Speleman F (2002) Elimination of primer-dimer artifacts and genomic coamplification using a two-step SYBR green I real-time RT-PCR. Anal Biochem 303:95–98
Varga A, James D (2006) Real-time RT-PCR and SYBR Green I melting curve analysis for the identification of Plum pox virus strains C, EA, and W: effect of amplicon size, melt rate, and dye translocation. J Virol Methods 132:146–153
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This work was supported with research funds (2008C22081) from the Science and Technology Bureau of Zhejiang Province and Zhejiang Natural Science Foundation (Y3090166), China.
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Cheng, J., Jiang, Y., Rao, P. et al. Development of a single-tube multiplex real-time PCR for detection and identification of five pathogenic targets by using melting-curve analysis with EvaGreen. Arch Virol 158, 379–386 (2013). https://doi.org/10.1007/s00705-012-1493-6
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DOI: https://doi.org/10.1007/s00705-012-1493-6