检测PRV野毒株、PCV-2及PPV多重PCR方法的建立及初步应用

Establishment and application of a multiplex PCR assay for simultaneous detection of PRV wild-type strain,PCV2 and PPV

根据GenBank中的猪伪狂犬病病毒(PRV)gE、猪圆环病毒2型(PCV-2)ORF2、猪细小病毒(PPV)VP2基因序列,设计了3对引物,成功建立了检测PRV野毒株、PCV-2和PPV的多重PCR诊断方法,扩增产物分别为288 bp、419 bp、681 bp。敏感性、特异性试验结果显示,该PCR对3种病毒的最低核酸检测量分别为PRV 48.2 pg/L、PCV-2 36.7 pg/L、PPV 0.25 ng/L,而PRV(gE基因缺失株)、猪瘟病毒(CSFV)、猪繁殖与呼吸综合征病毒(PRRSV)、大肠杆菌的扩增结果均为阴性。对87份自然感染病猪样品的检测结果表明,该多重PCR检测结果与单一PCR检测结果完全符合。结果表明,该多重PCR方法具有很好的特异性和敏感性,可用于临床PRV野毒株和gE基因缺失疫苗株、PCV-2和PPV的检测。

According to the gene sequences in GenBank of porcine pseudorabies virus（PRV） gE,porcine circovirus type 2（PCV2） ORF2 and porcine parvovirus（PPV） VP2,three pairs of specific primers were designed for amplifying the three specific fragments of PRV,PCV2 and PPV,respectively.After optimization of annealing temperature and primers concentrations,a multiplex PCR assay（mPCR） was established for simultaneous detection of the three viruses.Three specific bands,288 bp（PRV gE）,419 bp（PCV-2） and 681 bp（PPV）,were obtained.The sensitivity and specificity tests showed that the mPCR was highly sensitive and as little as 48.2 pg/L PRV,36.7 pg/L PCV-2 and 0.25 ng/L PPV could be detected.No specific bands were amplified from other viruses and bacteria by the mPCR.Eighty-seven clinical samples were detected by the mPCR.The results revealed that the established PCR assay was sensitive,specific and reproducible,and could be used for the rapid detection of PRV,PCV-2 and PPV.