检测PCV_2、PPV、PRV疫苗株与野毒株的多重PCR方法

Multiplex PCR for Rapid Detection of Pseudorabies Virus, Porcine Parvovirus and Porcine Circovirus Type 2

本文建立了一种同时检测猪圆环病毒2型(PCV2)、细小病毒(PPV)、及伪狂犬病毒(PRV)疫苗株与野毒株 的多重PCR方法。根据GenBank上发表的PCV2、PPV和PRV gB、gE基因序列,针对各自保守区各设计一对特 异性引物,用这四对引物对同一样品中的PCV2、PPV和PRV gB、gE进行检测,结果可同时扩增出269bp(PCV2)、 581bp(PPV)、372bP(PRV gB)及147bp(PRV gE)四条特异性片段。对JEV、PRRSRV、大肠杆菌和双蒸水的PCR 扩增结果均为阴性;敏感性测定结果表明,该多重PCR能检出10pg PCV2、PPV和PRV gB、gE检测敏感度分别为 106.2、103.8、10-5.8 TCID50的模板。该方法的建立对临床上进行这三种疾病的鉴别诊断和混合感染的检测具有重要 意义。

A multiplex PCR（mPCR） assay was developed and evaluated for its effectiveness as a means to simultaneously detect multiple viral infection of swine . Specific primers for each of the three common DNA viruses, Pseudorabies virus （PRV）, Porcine parvovirus （PPV） and Porcine circovirus type 2（PCV2） were used to test the procedure,Four specific bands of 269bp（PCV2）, 583 bp（PPV） 372 bp（PRV gB）and147 bp（PRV gE）were amplified. The assay proved to be sensitive when a composite of all three viruses were amplified, including both field and gene-deleted permutations of PRV. No specific band was amplified from other pathogenic viruses and bacteria. As little as 10 pg PCV2, 10^-6.2 TCID50 PPV,10^-3.8TCID50／PRV gB and 10^-5.8 TCID50 PRV gE were detected in this mPCR. This method could effectively detect infection of PCV2 ,PPV,field and gene-deleted permutations of PRV from clinical samples.