委内瑞拉马脑炎病毒一步法荧光定量RT-PCR方法的建立

Establishment of a One-Step Real-Time RT-PCR Method for the Detection of Venezuelan Equine Encephalitis Virus

委内瑞拉马脑炎(Venezuelan equine encephalitis,VEE)是由委内瑞拉马脑炎病毒(Venezuelan equine encephalitis virus,VEEV)复合物引起的一种人兽共患病。我国目前尚无该病,因此建立一种快速准确的检测方法对预防和控制该病至关重要。本研究通过分析委内瑞拉马脑炎病毒nsp1基因序列的保守性,设计了一对能检测VEEV复合物所有亚型毒株的特异性引物和Taqman探针。通过反应体系和反应条件优化,建立了检测委内瑞拉马脑炎病毒复合物的一步法实时荧光定量RT-PCR方法。该方法检测VEEV经体外转录获得的RNA,检测的灵敏度达3.27×102拷贝/μL。以该方法检测与VEEV同属的东方马脑炎病毒(EEEV)和西方马脑炎病毒(WEEV)nsp1基因相同区段的RNA以及同属的基孔肯雅病毒(CHIKV),结果均为阴性,表明该方法具有良好的特异性。该方法将为可能在我国出现的委内瑞拉马脑炎的侦检提供有效技术手段。

Venezuelan equine encephalitis（VEE）is a zoonotic disease caused by the Venezuelan equine encephalitis virus（VEEV）complex.This disease has not yet been reported in China,and it is therefore essential to establish a rapid and accurate method for detection of the virus in order to prevent and control this disease.In this study,a one-step real-time quantitative RT-PCR method was developed for the detection of the VEEV complex.A pair of specific primers and a Taqman probe were designed corresponding to a conserved region of the VEEV gene nsp1,allowing the detection of all known strains of different subtypes of the virus.Using RNA synthesized by in vitro transcription as template,the sensitivity of this method was measured at 3.27×102 copies/μL.No signal was generated in response to RNA from Chikungunya virus（CHIKV）,nor to RNA encoding the nsp1 fragment of Eastern equine encephalitis virus（EEEV）or Western equine encephalitis virus（WEEV）,all of which belong to the same genus as VEEV.This indicates that the method has excellent specificity.These results show that this one-step real-time quantitative RT-PCR method may provide an effective tool for the detection of VEEV in China.