Development of Multipex PCR for detection of Porcine Pseudorabies, Porcine Parvovirus and Japanese Encephalitis virus

According to sequences of PRV ,PPV and JEV obtained from GenBank,three pairs of primers were designed by biosoftware Arraydesigner 2.0. Nucleic acids of PRV Fa ,PPV B1 and SA14-14-2 strains were extracted from challenged BHK-21 cell 、IBRS cell and Vero cell respectively. Mixed templates of cDNA of JEV and DNA of PRV, PPV were amplified. The Multiplex PCR was developed and reaction conditions were optimized. The specified amplification fragments of PRV were 324 bp, PPV 471 bp and JEV 238 bp, respectively. Positive amplified fragments were obtained from PRV Fa, PRV Chongqing A, 8F20, BUK, Germany Bartha-K61, PRV Ea, PRV vaccine, PPV B2, PPV SR-1,SR-2, SR-3, JEV SA14-14-2 strains. DNA fragments were not amplified from cultural cells challenged with PRRSV、CFSV、PCV-2 and control cultural cells. The sensitivity of multiplex PCR for PRV, PPV DNA and JEV cDNA reached 17.5 pg,13.7 pg and 20 pg,respectively.This method is suitable for simultaneous diognosis or differentiation of PRV, PPV and JEV.