荧光恒温扩增技术检测肠道病毒71型方法的建立与评价

doi:10.3969/j.issn.1673-8640.2014.11.009

检验医学 ›› 2014, Vol. 29 ›› Issue (11): 1115-1119.DOI: 10.3969/j.issn.1673-8640.2014.11.009

荧光恒温扩增技术检测肠道病毒71型方法的建立与评价

曹凌峰，苏犁云，董妞妞，徐锦

复旦大学附属儿科医院临床检验中心,上海 201102

收稿日期:2014-04-18 出版日期:2014-11-01 发布日期:2014-11-28
作者简介:曹凌峰，男，1972年生，硕士，副主任技师，主要从事病毒学检验及机制研究。
基金资助:
上海市公共卫生优秀学科带头人计划(GWDTT201221)

Establishment and evaluation of fluorescence isothermal amplification assay for the detection of Enterovirus 71

CAO Lingfeng, SU Liyun, DONG Niuniu, XU Jin.

Clinical Laboratory Center, Children's Hospital of Fudan University, Shanghai 201102, China

Received:2014-04-18 Online:2014-11-01 Published:2014-11-28
Contact: 徐锦,联系电话:021-64931893。

摘要/Abstract

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。

关键词: 肠道病毒71型, 荧光恒温扩增技术, 手足口病, 敏感性, 特异性

Abstract:

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 199 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 119 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 100% 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.

Key words: Enterovirus 71, Fluorescence isothermal amplification assay, Hand, foot, mouth disease, Sensitivity, Specificity

中图分类号:

R446.1

曹凌峰，苏犁云，董妞妞，徐锦. 荧光恒温扩增技术检测肠道病毒71型方法的建立与评价[J]. 检验医学, 2014, 29(11): 1115-1119.

CAO Lingfeng, SU Liyun, DONG Niuniu, XU Jin.. Establishment and evaluation of fluorescence isothermal amplification assay for the detection of Enterovirus 71[J]. , 2014, 29(11): 1115-1119.

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荧光恒温扩增技术检测肠道病毒71型方法的建立与评价

Establishment and evaluation of fluorescence isothermal amplification assay for the detection of Enterovirus 71

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