摘要
通过RT-PCR方法克隆了H3亚型猪流感病毒HA基因一段靶序列，构建重组质粒作为标准阳性模板。根据GenBank中的H3亚型猪流感病毒HA基因保守序列设计了用于FQ-PCR的1对引物和1条TaqMan探针。通过条件优化，以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，建立了检测H3亚型猪流感的荧光定量PCR方法。结果表明，该方法检测灵敏度可达1．0×10^0拷贝／μL，线性范围为10^9-10^0，达10个数量级；对起始浓度为1．0×10^9、1．0×10^8、1．0×10^7拷贝／μL的标准品的最终实际测得值（Ct）分别为13．68，18．21和20．57；变异系数分别为0．31％、0．17％和0．12％，均小于5％，说明此方法具有良好的准确性和重现性。对阳性组织病料的检测表明，该方法的检测灵敏度高出常规PCR，与套式PCR具有相近的灵敏度。
The part sequences of HA segment of SIV were cloned into pMD-18 T vector and the recombinant plasmid was constructed and used as standard positive template for real time PCR. Meanwhile, according to the published HA segment of SIV sequences of SIV in GenBank, a pair of primers and a TaqMan probe were designed. The FQ-PCR assay was conducted by quantitative concentration of serial 10 fold dilutions of recombinant plasmid DNA by optimizing circulation parameters. A standard curve was achieved and the result showed that the sensitivity of this method was10^9-10^0copy/μL and the linear relation was excellent. Meanwhile, the final measure values on initiative concentrations of 1.0×10^9、1.0×10^8、1.0×10^copy/μL DNA were 13.68,18. 21 and 20. 57;and the coefficient of variation was 0. 31% ,0. 17% and 0. 12%, respectively, all below 5%. Furthermore, the sensitivity of FQ-PCR was roughly equal to that of nPCR and was higher than that of PCR by detecting the positive samples.
出处
《动物医学进展》
CSCD
北大核心
2009年第6期30-35,共6页
Progress In Veterinary Medicine
