Development A Multiplex PCR For Rapid Detection Of Porcine Pseudorbies Virus, Porcine Parvovirus And Porcine Circovirus Type 2

1. Three pairs of primer were designed for amplificaton of porcine pseudorbies virus (PRV) gB, gE, and TK gene in the same multiplex PCR (multi-PCR) reaction for differentiation of vaccine strains and field isolates. The result showed that three specific bands at the expected size were obtained, including 427 bp (gB gene), 298 bp (gE gene), and 208 bp (TK gene), respectively. Then four different gene deletional vaccines of PRV were detected by the multi-PCR. One expected specific band was observed in one of vaccines , while two bands in the others. The detection results showed that the multi-PCR has high sensitivity and specifity, and should be applied pathogen diagnosis and epidemiological investigation in the future.2. Five pairs of specific primers were designed according to the published sequences of Porcine circovirus type 2 (PCV2), Porcine parvovirus (PPV), Pseudorabies virus (PRV) in GenBank. The single PCR of PCV2, PPV, PRV was established,and the fragments of DNA amplification appeared in 657bp(PPV),490 bp(PCV2),372 bp(PRV-gB) and 298bp(PRV-gE), respectively. Based on the optimized single PCR conditions, the multiplex PCR of PRV-PCV2-PPV was developed. It provided a rapid, sensitive and specific detection technique for the prevention and control of PRV, PCV2 and PPV infection.3. To improve the amplification of Porcine pseudorabies virus gB gene , the effects of DMSO with different concentration on the PCR Amplification of GC-rich gB gene investigated. The results showed that the specifcity for PCR is increased in the presence of DMSO, The gB gene best amplified by adding 4%～10% DMSO into PCR reaction solution in GC-rich Porcine Pseudorbies virus Genome DNA template.