盖塔病毒TaqMan探针荧光定量RT-PCR检测方法的建立

TaqMan-based real-time RT-PCR for rapid detection of the Getah virus

为了建立特异、灵敏、快速的荧光定量RT-PCR方法用于检测盖塔病毒(Getah virus,GETV)的核酸,并初步应用于GETV的临床标本检测。根据GenBank登录的GETV全基因组序列,应用生物学软件进行序列比对,在NSP1保守区设计1对特异性引物和TaqMan探针。建立实时荧光定量RT-PCR检测GETV的方法,并对建立的方法进行敏感性、特异性测定和临床样品检验。结果显示,该方法能特异性地鉴别检测GETV;检测灵敏度高,检测的最低模板量可以达到2.03×10^1 copies/μL;将收集到的10000只蚊子样品分成100份进行检测,共检测出8份GETV阳性样品,与常规反转录PCR(RT-PCR)检测及病毒分离结果吻合。本研究建立的GETV TaqMan实时荧光定量RT-PCR检测方法特异、灵敏,适用于GETV的临床早期诊断。

To establish a TaqMan-based real-time reverse transcription-polymerase chain reaction(RT-PCR)for detection of the Getah virus.The established method was used primarily to test clinical samples for GETV.The gene sequences of GETV were downloaded from GenBank.They were aligned using biologic software,and specific primers and probes were designed in the conserved region of the NSP1 gene for the GETV.The primers,probes and reaction conditions were optimized to improve the sensitivity and specificity of real-time RT-PCR.Clinical specimens collected from Culex tritaeniorhynchus underwent real-time RT-PCR.The specificity of the RT-PCR was high,with no cross reactions with AKAV,BATV,JEV,PRRSV,PRV,PCV2,CDV or CPV.The sensitivity of realtime RT-PCR was 2.03×10^1 copies/μL,and viral RNA could be detected directly from clinical specimens.It took 3-4 h to complete real-time RT-PCR.8 positive samples were detected for GETV in 100 samples of mosquitoes,coincide with the the traditional virus isolation and identification.This TaqManTM-based real-time RT-PCR was a rapid,sensitive and specific method for the molecular diagnosis of the GETV.