Establishment And Application Of A Multiplex RT-PCR Assay For Simultaneous Detection Of The North American Genotype And The European Genotype Of Porcine Reproductive And Respiratory Syndrome Virus(PRRSV)

In order to establish one assay to simultaneous and differential detection of the North American (NA) genotype and the European (EU) genotype of porcine reproductive and respiratory syndrome virus (PRRSV), three pairs of specific primers were designed. They were named as CO-ORF7, EU-ORF5and AM-Nsp2, respectively, of which CO-ORF7specifically amplified433bp/398bp ORF7gene fragment of the NA genotype and the EU genotype, AM-Nsp2specifically amplified338bp or248bp Nsp2gene fragment of the classical strain or the highly pathogenic (HP) variant of the NA genotype, and EU-ORF5specifically amplified614bp ORF5gene fragment of the EU genotype. After optimization of the reaction conditions, one multiplex RT-PCR detection method of PRRSV was successfully established. The assay could only react with PRRSV, but not with other important porcine pathogens such as classical swine fever virus (CSFV), foot-and-mouse disease virus (FMDV), porcine circovirus type2(PCV2), porcine pseudorabies virus (PRV) and porcine parvovirus (PPV). The detection limit was as little as1.67×103copies/μL of the correlative recombinant plasmids. The established assay were successfully used to detect PRRSV in176clinical suspected samples, and45samples were positive for PRRSV, of which41samples belonged to HP-PRRSV,4samples belonged to classical PRRSV and the EU genotype strains could not be found. Since clinical co-infection of CSFV and PRRSV are common, to rapid detection of CSFV and PRRSV, three pairs of specific primers named CSF-NS2, AM-Nsp2and EU-oRF5respectively were designed. CSF-NS2specifically amplified508bp NS2gene fragment of CSFV, AM-Nsp2specifically amplified338bp or248bp Nsp2gene fragment of the classical strain or the highly pathogenic (HP) variant of the North American (NA) genotype PRRSV (NA-PRRSV), and EU-ORF5specifically amplified614bp ORF5gene fragment of the European (EU) genotype PRRSV (EU-PRRSV). After optimization of the reaction conditions, a multiplex RT-PCR assay was established for simultaneous and differential detection of CSFV, the classical strain and HP variant of NA-PRRSV and the strain of EU-PRRSV. The assay could only react with CSFV and PRRSV, but not with FMDV, PCV2, PRV and PPV. The detection limit of the method was as little as1.67×103copies/μL of each of the four recombinant plasmid templates. The established assay were successfully used to detect CSFV and PRRSV in106clinical samples, of which4samples were positive for CSFV and PRRSV,7samples for CSFV and17samples for HP-PRRSV.This study successfully established two methods for detection of PRRSV, of which one was the multiplex RT-PCR assay for simultaneous and differential detection of the NA genotype and the EU genotype PRRSV, the other was the multiplex RT-PCR assay for simultaneous and differential detection of CSFV and the NA genotype and the EU genotype PRRSV. They were suitable for clinically rapid detection and epidemic surveillance of PRRSV and CSFV.