狂犬病病毒实时荧光定量PCR检测方法的建立和阳性对照的制备

摘要

Objective Establish the fluorescent quantitative RT-PCR detection method for rabies virus (RV) and construct Rnase-resistant virus-like particles as positive controls. Methods Analyze the database in GenBank,the probe and the primers were designed in the conservative region of N gene of rabies virus and the method of real-time fluorescent quantitative PCR was obtained;On the basis of MS2 phage,with the technology of gene recombination,prepare the Rnase-reslstant virus-like particles for RV positive controls; Results Rnase-resistant vires-like particles were obtained after prokaryotic expression in E. Coli. The designed primers and probe were confirmed to be very specific and conservative ,and be sensitive to concentration of 15 copies/μl. Conclusion Estabhshed the method of detecting rabies virus by reverse transcription real-time quantitative PCR,obtained the Rnase-resistant and no infectivity virus-like particles as positive controls of rabies virus.%目的 建立狂犬病病毒实时荧光定量PCR检测方法,制备狂犬病病毒(rabies virus,RV)的假病毒颗粒阳性对照.方法 在RV的N基因保守区设计引物和探针,建立反转录实时荧光定量PCR检测方法,克隆得到噬菌体MS2的装配蛋白和壳蛋白基因(MS2),将其重组到质粒pET-28b(+)中,再将RV的N基因片段重组到MS2下游,经原核表达得到RV假病毒颗粒.结果 实时荧光定量PCR检测RV重组质粒最低检测限为15拷贝/μl,重组表达质粒经原核表达可形成耐RNase的RV病毒样颗粒.结论 建立了反转录实时荧光定量PCR检测RV核酸的方法,成功构建了可耐受RNase且无传染性的RV阳性对照.