荧光定量RT-PCR检测猪日本脑炎病毒

Detection of JEV by Fluorescent Quantitative PCR

通过RT-PCR方法克隆了日本脑炎病毒(JEV)N基因片段序列,构建重组质粒作为标准阳性模板,同时根据GenBank中的靶序列设计了荧光定量RT-PCR的1对引物和1条TaqMan探针。通过条件优化,以定量的10倍系列稀释的质粒为标准品进行荧光定量PCR扩增并制作标准曲线,建立了JEV检测的荧光定量PCR方法。结果表明,该方法检测灵敏度可达1.0×100拷贝/μL,线性范围为109～100,达10个数量级;对起始浓度为1.0×109、1.0×108和1.0×107拷贝/μL的标准品的最终实际测得值(Ct)分别为17.33、20.52和23.90,变异系数分别为0.54%、0.74%和0.53%,均小于5%。对阳性组织病料的检测表明该方法的灵敏度高出常规PCR,与套式PCR(nested-PCR,nPCR)具有相近的灵敏度。

The part sequences of JEV E gene was cloned into pMD18 T vector and the recombined plasmid was constructed and used as standard positive template.Meanwhile,according to the published E gene sequences of JEV in GenBank,a pair of primers and a TaqMan probe were designed.The FQ-PCR assay was carried out by quantitative concentration of serial 10 fold dilutions of recombined plasmid DNA by optimizing circulation parameters.A standard curve was achieved and the result showed that the sensitivity of this method was 1.0×10^0copy/μL and the linear relation was excellent.Meanwhile,the final measure value on initial concentration of 1.0×10^9,1.0×10^8,1.0×10^7copy/μL DNA was 17.33,20.52 and 23.90 respectively;and the coefficient of variation was 0.54%,0.74% and 0.53%,respectively,all below 5%.Furthermore,we found that the sensitivity of FQ-PCR was roughly equal to that of nPCR and was better than that of PCR by detecting the positive field samples.