Establishment And Intial Application Of Multiplex PCR Assay For Detecting Six Swine Viruses

Classical swine fever virus (CSFV), Porcine reproductive and respiratory syndromevirus, Japanese encephalitis virus (JEV),Porcine circovirus type2(PCV-2), Porcinepseudorabies virus (PRV) and Porcine parvovirus (PPV) were the major pathogen which canarose respiratory symptoms and reproductive failure and causes a great economic lose toswine production. When infections cause abortion and/or stillbirth, determining whether thecausal agents such as CSFV, PRRSV, JEV, PPV, and PRV or multiple viruses are difficultbased only on clinical signs. The traditional methods for diagnosis of viral diseases such asviral isolation or serological diagnostic methods are time consuming and low sensitivity, thusthess assays couldn’t meet the demand of clinical diagnosis. As the multiplex RT-PCR andthe multiplex PCR chould detect and to differentiate simultaneously multiple viruses in asingle sample on the basis of amplicon size, Multiplex PCR was widely used in detection ofviral infection.In this study, six pairs of targeting six viral conservative gene were synthetized.Themultiplex PCR reaction condition was optimized and the multiplex PCR for simultaneouslydetecting PCV-2, PPV, PRV, CSFV, PRRSV and JEV was established. Some results were asfollows:1. Foundation and Initial Application of Multiplex RT-PCR Method for detecting CSFV,PRRSV and JEV infectionThree pairs of primers were designed and synthesized according to the highly conservedregions of CSFV, PRRSV and JEV genome sequence in Genbank. The multiplex RT-PCRreaction condition was optimized and the multiplex RT-PCR method for detecting CSFV,PRRSV and JEV infection was established. The results showed great specificity andreproducibility. Also, the assay can detecting the level of pg viral RNA.The initialamplication test showed52clinical samples were subjected to RT-PCR.2. Establishment and Initial Application of Multiplex PCR assay for detecting PCV-2,PPV and PRV infectionAccording to the highly conserved genome sequence of PCV-2, PPV and PRV, three pairsof primers targeting PCV-2ORF2，PPV NS1and PRV gB were synthetized. The multiplexPCR reaction condition was optimized and the multiplex PCR for simultaneously detectingPCV-2, PPV and PRV was established. The specificity test showed that a fragment of351bp, 265bp and198bp was amplified from the genomic DNA of PCV-2, PPV and PRVrespectively, no amplification was achieved from negative control groups. the assay candetecting the level of pg viral DNA. The initial application test showed that the thirty-nineclinical samples were subjected to multiplex PCR. Among these samples, PCV-2positivepercentage was53.8%.3. Development of multiplex PCR for simultaneous detection of six swine DNA andRNA virusesAbove on multiplex PCR assay for detecting PCV-2, PPV and PRV and multiplexRT-PCR assay for detecting CSFV, PRRSV and JEV, The multiplex PCR reaction conditionwas optimized and the multiplex PCR for simultaneously detecting PCV-2, PPV, PRV, CSFV,PRRSV and JEV was established. The assay was highly specific in detecting one or more ofthe same viruses in various combinations in specimens. The multiplex PCR assay was alsoshown to be sensitive because it could detect at least450pg of viral genomic DNA or RNAfrom a mixture of six viruses in a reaction. Thirty-nine clinical samples were deteced bymultiplex PCR,25.6%of the samples were co-infected with PCV-2and PRRSV indicatedthat PCV-2and PRRSV have a high prevalence in Shaanxi province.