埃可病毒9型荧光RT-PCR快速检测方法的建立及应用  

TaqMan^(TM)-based Real-time RT-PCR for Rapid Detection of the Echovirus 9 Serotype

作　　者：邱晓枫[1] 张国忠[1] 黄志成[1] 潘劲草[1] 于新芬[1] 

机构地区：[1]杭州市疾病预防控制中心,杭州310021

出　　处：《病毒学报》2017年第6期836-841,共6页Chinese Journal of Virology

基　　金：杭州市科技发展计划项目(项目号:20120633B22);题目:埃可病毒9型的快速检测方法建立以及流行特征研究;杭州市农业与社会发展科研计划项目(项目号:20170533B76);题目:杭州市区手足口病患儿中埃可病毒的检测及其分子特征研究~~

摘　　要：为了建立特异、灵敏、快速的荧光定量RT-PCR方法用于检测埃可病毒9型病毒核酸,并初步应用于埃可病毒9型的临床标本检测。根据GenBank登录的埃可病毒9型毒株VP1基因序列,应用生物学软件进行序列比对,在保守区设计特异性引物和TaqMan探针。对反应条件进行优化,验证方法的特异性、灵敏度和重复性,同时对疑似埃可病毒9型病例标本进行检测。该方法对埃可病毒9型的检出有高度特异性,与EV71、CA16、CA24v、CA6、CA10、埃可病毒30型等均无交叉反应,检测灵敏度均达0.1TCID50/mL,可从疑似埃可病例粪便标本中直接检测病毒核酸,检测仅需3~4h。本研究建立的埃可病毒9型TaqMan荧光定量RT-PCR检测方法特异、灵敏,适用于临床早期诊断。To establish a TaqManXM-based real-time reverse transcription-polymerase chain reaction （RT- PCR） for detection of the Echovirus 9 serotype. The established method was used primarily to test clinical samples for Echovirus 9. The gene sequences of Echovirus 9 were downloaded from GenBank. They were aligned using biologic software, and specific primers and probes were designed in the conserved region of the VP1 gene for the Echovirus 9. The primers, probes and reaction conditions were optimized to improve the sensitivity and specificity of real-time RT-PCR. Clinical specimens were collected from an epidemic of viral encephalitis in children underwent real-time RT-PCR. The specificity of the RT-PCR was high , with no cross reactions with EV71, CA16, CA24v, CA6, CA10, ECHO-6 or ECHO-30. The sensitivity of real- time RT-PCR was 0.1TCID50/mL, and viral RNA could be detected directly from clinical specimens. It took 3-4 hours to complete real-time RT-PCR. This TaqManTM-based real-time RT-PCR was a rapid, sensitive and specific method for the molecular diagnosis of the Echovirus 9 serotype.

关 键 词：埃可病毒9型(ECHO-9) 荧光定量RT-PCR TAQMAN探针 

分 类 号：R373.24[医药卫生—病原生物学]