用多重PCR鉴别猪伪狂犬病野毒与疫苗毒的研究

Detection of porcine pseudorabies virus and differentiation of natural PRV from vaccinal PRV by multi-PCR

根据基因库中的猪伪狂犬病病毒(PRV)各基因的序列,设计了与PRV的 gB、gD、gE基因序列互补的3对引物。对样品中的PRV DNA模板进行了多重PCR扩增及反应条件的优化,结果同时得到与设计相符合的3条特异性条带,分别为549 bp(gB)、429 bp(gD)、366 bp(gE)。用这3对引物对三基因缺失疫苗毒的样品DNA模板进行多次多重 PCR扩增,均能稳定得到与设计相符合的2条特异性条带。敏感性试验结果表明,多重 PCR可以检测到 106 pg三基因缺失疫苗毒或756 pg PRV野毒的核酸模板量。特异性试验结果表明,以正常对照细胞及猪圆环病毒和猪细小病毒DNA为模板进行多重PCR扩增,均无任何条带。

A multiplex polymerase chain reaction was developed and optimized to detect simultaneously porcine pseudorabies virus (PRV) and differentiate natural PRV from vaccinal TK^(-)/gI^(-)/gE^(-)PRV. Three pairs of specific primers were designed according tothe conservative sequences of PRV gB,gD and gE genes in GenBank. All samples containing PRV and recombinant PRV were amplified desirably by the multiplex PCR using the three sets of primers and then the multiplex PCR conditions were optimized. Three expected specific bands of PRV gB gene 549bp, gD gene 429bp and gE gene 366bp in length with the templates as little as 756pg of PRV or 106pg of vaccinal TK^(-)/gI^(-)/gE^(-) PRV were obtained by the multiplex PCR.The specific experiment showed that the multiplex PCR is qualified specifically to detect natural PRV and vaccinal TK^(-)/gI^(-)/gE^(-)recombinant PRV.