荧光恒温扩增技术检测肠道病毒71型方法的建立与评价

摘要

目的：利用荧光恒温扩增（SAT）技术建立一种快速可靠的肠道病毒71型（EV71）检测方法。方法通过设计特异性的 EV71 RNA 扩增引物及优化探针技术，使用 M-MLV 反转录酶及 T7 RNA 多聚酶对 EV71 RNA进行核酸扩增，同时利用荧光定量聚合酶链反应（PCR）仪进行实时的荧光信号收集和检测。检测复旦大学附属儿科医院儿童手足口病患儿粪便样本199例，以 EV71型核酸检测试剂盒作参比方法，DNA 测序为第三方验证方法，对研究数据进行 Kappa 一致性分析。结果SAT 技术共检测到119例 EV71阳性样本，其中21例荧光探针法检测为阴性，经测序证实20例样本仍为阳性。SAT 与荧光探针法检测结果的 Kappa 值为0．789。SAT 方法的敏感性100％、特异性98．77％。结论本研究建立的 EV71型 RNA SAT 技术具有敏感性高、特异性强、准确、快速可靠等优点，适用于临床对 EV71感染的快速诊断。%Objective To establish a rapid and reliable fluorescence isothermal amplification assay (SAT)for the detection of Enterovirus 71 (EV71 ).Methods By designing specific primers and optimal probe and using M-MLV reverse transcriptase and T7 RNA polymerase,the SAT was developed for detecting EV71 based on isothermal RNA amplification and fluorescence quantitation polymerase chain reaction (PCR).A total of 1 99 stool specimens from the children with hand,foot,mouth disease from Children′s Hospital of Fudan University were determined.Using EV71 PCR fluorescence diagnostic kits as reference kit and DNA sequencing as the third party verification method,the Kappa analysis was performed on the study data for consistency.Results A total of 1 1 9 stool specimens were positive by SAT, and there were 21 specimens being negative by PCR-fluorescence probe assay,but 20 specimens were confirmed to be positive by DNA sequencing.The Kappa value between SAT and PCR-fluorescence probe assay was 0.789.The sensitivity and specificity of SAT were 1 00% and 98.77%,respectively.Conclusions This study indicates that RNA SAT for the detection of EV71 is a sensitive,specific,accurate,rapid and reliable method,and it should be a potential method for the rapid detection of EV71 infection.