猪瘟病毒野毒株与疫苗株双重SYBR GreenⅠ实时荧光定量PCR检测方法的建立

Development of a SYBR GreenⅠreal-time PCR assay to differentiate China wild-type and vaccine strain of classical swine fever virus

为建立猪瘟病毒(CSFV)兔化弱毒疫苗(C株)与野毒株的快速鉴别检测方法,本研究根据GenBank登录的CSFV C株与野毒株的E2基因序列差异,设计2对特异性引物,建立了同时检测C株与野毒株的双重SYBR GreenⅠ实时荧光定量PCR方法。应用该方法对CSFV野毒和疫苗株的最低检出量为10拷贝;检测牛病毒性腹泻病毒Ⅰ型、猪繁殖与呼吸综合征病毒、伪狂犬病病毒和猪圆环病毒2型4种病毒均呈阴性;批内、批间重复试验的变异系数均低于3%。应用该方法检测从黑龙江省各地区猪场采集的600份样品,结果表明野毒株的阳性率为30.5%。该方法可应用于CSFV野毒株和C株的鉴别检测。

In this study,a duplex SYBR Green Ⅰ real-time PCR for differentiating wild-type and C-strain of CSF virus（CSFV） was established.Two pairs of primers were designed according to the differences of E2 gene sequences between wild-type and C-strain in GenBank.This assay could specifically differentiate CSFV from Bovine viral diarrhea viruses type 1,Pseudorabies virus and Porcine circovirus type 2.The assay was able to detect 10 copies for wild-type and C-strain with high reproducibility.Using the duplex real-time RT-PCR,600 clinic samples of pigs were analyzed for porcine wild-type CSFV and 30.5% were positive.