Simultaneous Detection Of Six Swine Viruses By An EvaGreen Multiplex Real-time PCR

Porcine circovirus virus Ⅱ type (PCV2), porcine parvovirus (PPV), porcine reproductiveand respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), Japaneseencephalitis virus (JEV) and pseudorabies virus (PRV) all can cause similar clinical respiratoryand/or reproductive disorder symptoms and often showed mixed infection, which led tosignificant losses to the pig industry. Conventional etiology and serological detection methodswere difficult to simultaneously distinguish these diseases, as well as operation complicated,time-consuming and less sensitive. Especially, when coinfected with some pathogens, it wasdifficult for early detection and prone to erroneous judgement. It was, therefore, significant todevelop an effective and reliable approach for epidemiological surveillance and diseasemanagement. A multiplex real-time PCR thus can be applied to rapid detection of mixedinfection of pathogens.According to the principle of fluorescence group, multiplex real-time PCR assays aregenerally classifed into fuorescent-probe-based assay and fuorescent-dye-based assay.Fluorescent probes-based assay is high specificity, but compared with dye-based assay,probe-based assay is expensive, and it is not able to simultaneously detect fve viruses due to therestriction from real-time PCR instrument. Dye-based multiplex real-time PCR could satisfy tosimultaneously detect more than five targets using melt curve analysis. The widely usedfluorescent-dye is SYBR Green Ⅰ (SG), but there are problems to detect some PCR productsdue to its preferential binding to amplicons with higher G+C%and larger size in multiplexassays, thus making it difficult to insure similar or equal amplification efficiency betweendifferent templates. EvaGreen (EG), a new saturated dye that intercalated every single nucleotideof the double-stranded DNA, has been shown to be more suitable than SG for multiplexreal-time PCR because of highly sensitive and extremely stable. Therefore, an EvaGreenmultiple real-time PCR was establised to multiplex detection of six swine viruses with meltingcurve analysis in this study.To this end, optimization of a singleplex real-time PCR was essential for the subsequentmultiplex real-time PCR. Melt peaks corresponded to amplificons from six swine viruses insingleplex real-time PCR should be distinguished effectively, which was the premise to optimizesingleplex real-time PCR. Through targets and non-targets amplification using the selectedprimers, singleplex real-time PCRs were showed to be specific for PCV2, PPV, PRRSV, CSFV,JEV and PRV, respectively. The detection limit of the singleplex real-time PCRs could reach10 copies/μl for PCV2and CSFV,25copies/μl forPRV and JEV,50copies/μl forPPV and PRRSV.Serial concentrations of plates were detected, and reproducibility for both intra-assay andinter-assay all varied within5%. On the basis of optimization of the singleplex real-time PCRs,melting peak of amplicons for differentiation of six swine viruses was established with Tm valueof77.7±0.29°C,79.5±0.36°C,82.2±0.30°C,85.3±0.35°C,87.3±0.31°C and91.3±0.25°C forPCV2, PPV, PRRSV, CSFV, JEV and PRV, respectively. The results from targets and non-targetsamplification showed that the multiplex EvaGreen real-time PCR system was specific for PCV2,PPV, PRRSV, CSFV, JEV and PRV. For sensitivity detection of six swine viruses, the detectionlimit of PCV2, PRRSV and JEV was100copies/μl, PPV and CSFV250copies/μl, PRV500copies/μl. The multiplex assay is highly repeatable with both intra-assay and inter-assay varyingwithin5%. Considering mixed infection is usual in the natural setting, several co-infectioncombinations were chosen to detect sensitivity of the assay. The sensitivity of PCV2was250copies/μl in coinfections of CSFV, PPRSV up to250copies/μl in dual infections of PCV2andPRRSV, PCV2up to500copies/μl in dual infections of PCV2and PPV, PRRSV up to500copies/μl in triple infections of PCV, PPV and PRRSV. All these data presented a goodspecificity, sensitivity, repeatability of the multiple EvaGreen real-time PCR.Fifty-eight serum and sixty tissue samples were tested by the EvaGreen multiplex real-timePCR. In the test of serum samples, positive rate of PCV2was19.0%, PRRSV was24.1%, andmixed infection was10.3%. In the test of sixty tissue samples, the detection rate of PCV2, PPV,PRRSV and PRV was23.3%,15%,16.7%and15%respectively, the mixed infection rate ofPCV2and PRRSV, PCV2and PPV, PCV2, PPV and PRRSV was6.7%,5%and5%respectively,while positive rate of PCV2was21.6%by conventional PCR. Compared to conventional PCR,the EvaGreen multiplex real-time PCR displayed higher sensitivity as well as time-saving andefficient. In conclusion, in this study an EvaGreen multiplex real-time PCR for detection anddifferentiation of PCV2, PPV, PRRSV, CSFV, JEV and PRV was established successfully. Itcould be an alternative for epidemiological surveillance of the six swine viruses.