Establishment and Application of Multiplex PCR Assay for Detection of Porcine Parvovirus,Pseudorabies Virus and Porcine Circovirus Type 2

Porcine parvovirus(PPV),pseudorabies virus(PRV) and porcine circovirus type 2(PCV2) were three main viral pathogens of porcine reproduction barrier diseases.To identify and differentiate rapidly the cause(s) of clinical diseases,a multiple PCR/RT-PCR assay was developed.Based on the similarity of the viral sequences deposited in the GenBank database,the VP2 gene of PPV,the gD gene of PRV and the ORF2 gene of PCV2 were selected as the diagnostic targets.By using three pairs of virus-specific primers,three PCR/RT-PCR assay were established to amplify the conservative regions of the three viruses,respectively.Consequently,a multiplex PCR/RT-PCR method to detect the three viruses in one tube was developed.The multiple PCR/RT-PCR system would amplify a 313 bp fragment for PPV,a 217 bp for PRV and a 447 bp for PCV2 simultaneously or separately in the samples,depending on its infection status.By comparison,the test proved that the multiplex PCR method being 100% coincidence with the single PCR,and it could be used for this three virus detection and differential diagnosis.211 samples of pigs collected from 10 nosopoietic pig farms and outpatient cases were detected with the multiplex PCR assay established,the result showed that the positive rate of 42 positive PPV was 19.91%,the positive rate of 26 positive PRV was 12.32% and the positive rate of 56 positive PCV2 was 26.54%,more than,the mixed infection rate of 25 two kinds of mixed infection was 11.85%.It proved that the pigs had been infected by these three epidemics in Shanxi.