Establishment and application of a multiplex PCR assay for simultaneous detection of PCV2,PPV,PRV and PRRSV

According to the gene sequences in GenBank of porcine circovirus type 2(PCV2),porcine parvovirus(PPV),porcine pseudorabies virus(PRV) and porcine reproductive and respiratory syndrome virus(PRRSV),four pairs of specific primers were designed for amplifying the four specific fragments of PCV2 ORF2,PPV NS1,PRV gB and PRRSV N,respectively.After optimization of annealing temperature and primers concentrations,a multiplex PCR assay(mPCR)was established for simultaneous detection of the four viruses.The sensitivity and specificity tests showed that the mPCR was highly sensitive in that as little as 1 pg of the respective virus,29.0 pg(PCV2),17.3 pg(PPV),36.8 pg(PRRSV),38.4 pg(PRV).No band was amplified from classical swine fever virus(CSFV),swine influenza virus(SIV),porcine circovirus type 1(PCV1),bovine viral diarrhea virus(BVDV),Escherichia coli by the mPCR.119 clinical samples were detected by the mPCR.As a result,PCV2 was detected from all the samples,moreover,3 samples were positive for PPV,68 samples were positive for PRRSV.The multiplex method could be used to effectively detect and differentiate PCV2,PPV,PRV and PRRSV single or co-infection infected in clinical samples.