禽流感病毒H7N9型实时荧光定量RT-PCR检测方法的研究

目的 建立一种快速准确诊断禽流感病毒H7N9型的实时荧光定量RT-PCR检测方法。方法 参照国家生物技术信息中心(NCBI)公布的H7N9基因HA序列,设计特异性引物及TaqMan探针; 构建阳性重组质粒,验证其最低检测拷贝数、重复性及特异度。结果 试验构建的阳性重组质粒,线性关系在6×10² copies/μl～6×10⁸ copies/μl范围内检测结果良好; 用该方法测得最低拷贝数为600 copies/μl,特异度和重复性(CV<1%)良好,无交叉反应; 用该荧光定量RT-PCR检测方法检测8份阳性样品和8份阴性样品,准确率为100%(16/16)。结论 实验建立的实时荧光定量RT-PCR检测方法可用于禽流感病毒H7N9型的快速检测。

Devolepment of Real-time Fluorescence QuantitativeRT-PCR Detection for Avian Influenza Virus H7N9 Type

Objective To establish a rapid and accurate method of fluorescent quantitative PCR detection for Avian Influenza Virus H7N9 type.Methods A pair of primers and TaqMan fluorescent probes were designed specificly for H7N9 gene according to the published nucleotidesequence from National Center for Biotechnology Information.Positive recombinant plasmid was built and the minimum copies of detection,repeatability and specificitv were tested.Results The results showed that this assay obtained positive recombinant plasmid,the linear relation was fine in the range from 10² to 10⁸ copies/μl.The minimum copies was 500 copies/μl with this method,specificitv and repeatability(CV<1%)were fine,no cross reaction with other viruses.The precision was100%(16/16)with this method for detecting 8 positive samples and 8 negativesamples.Conclution The establishment methods of fluorescent quantitative PCR detection could be used for the rapid diagnosis Avian Influenza virus H7N9 types.