一步法荧光RT-PCR检测肠道病毒EV71的研究

Development of One-step Fluorescence RT-PCR for Detection of EV71 Enterovirus

目的建立一种快速准确诊断肠道病毒EV71型的实时荧光定量RT-PCR检测方法。方法参照NCBI公布的EV71基因序列,设计特异性引物及Taq Man探针;构建阳性重组质粒,验证其最低检测拷贝数、重复性及特异性。结果试验构建的阳性重组质粒,线性关系在9×10~2copies/μL～9×10~7copies/μL范围内检测结果良好;用该方法测得最低拷贝数为900 copies/μL,特异性和重复性(CV <5%)良好,无交叉反应;用该荧光定量RTPCR检测方法检测80份阳性样品和36份阴性样品,准确率为100%(116/116)。结论试验建立的实时荧光定量RT-PCR检测方法可用于肠道病毒EV71型的快速检测。

Objective To establish a rapid and accurate method of fluorescent quantitative PCR detection for En- terovims EV71 type. Methods A pair of primers and TaqMan fluorescent probes was designed specially for EV71 gene ac- cording to the published nucleotide sequence fi＇om National Center for BiotechnologT Information. Positive recombinant plas mid was built and the nlininmm copies of detection, repeatability and specificity were tested. Results The results showed that this assay obtained positive recombinant plasmid, the linear relation was fine in the range from 9 ×102 to 9 × 107copies/ μL. The minimum copies was 900 copies/IAL with this method, specificity and repeatability （ CV 〈 5 % ） were fine, no cross reaction with other viruses. The precision is 100% （116/116） with this method for detecting 80 positive samples and 36 neg- ative samples. Conclusion The establishment Methods of fluorescent quantitative PCR detection could be used for the rapid diagnosis EF71 Enteroviruses.