实时荧光定量PCR检测猪病料中的PRRSV与CSFV

Detection of PRRSV and CSFV Using Real-time PCR Assay

根据GenBank发表的PRRSV、CSFV基因核苷酸序列,应用Primer 5.0软件分别设计了2对特异性引物。以重组质粒作为阳性标准品,应用SYBR Green Ⅰ real-time PCR检测两种病毒的核酸含量。在样品稀释到10-1～10-11拷贝时,能得出良好的线性关系,相关系数为R2=0.999 8。用此法检测猪细小病毒(PPV)、伪狂犬病毒(PRV),结果均为阴性。建立了PRRSV、CSFV的标准曲线。该方法具有线性关系好、特异性强、敏感性高、重复性好等特点,为分析PRRSV、CSFV共感染猪体内病毒的含量提供了有效地检测方法。

According to the nucleotide sequences of porcine reproductive and respiratory syndrome virus（PRRSV） and classical swine fever virus（CSFV） in GenBank,two pairs of primers were designed and synthesized by using the primer 5.0 software.Real-time PCR was used to detect the load of two viruses using the recombinant plasmids as positive standard.A standard curve was achieved and the dynamic range was from 10-1-10-11 diluted times of the samples,and the dependent coefficient R2=0.999 8.There was no amplification with porcine parvovirus（PPV） and pseudorabies virus（PRV）.The results indicated that the real-time PCR showed an excellent linear relation,and was sensitive,specific,and repeatable.Therefore,the real-time PCR could be used as an effective tool for differentiating diagnosis of PRRSV and CSFV in epidemiological investigations.