8种脑炎相关虫媒病毒GeXP检测方法的初步建立  

Development of a GeXP Based Multiplex RT-PCR Assay for Simultaneous Detection of Eight Arboviruses Related to Encephalitis

作　　者：何玢[1] 王环宇[2] 张晨 王淼[2] 秦萌[3] 王克霞[1] 马学军[2] 

机构地区：[1]安徽理工大学医学院,淮南232001 [2]中国疾病预防控制中心病毒病预防控制所病毒基因工程国家重点实验室,北京102206 [3]北京市丰台区疾病预防控制中心,北京10071

出　　处：《病毒学报》2012年第1期57-62,共6页Chinese Journal of Virology

摘　　要：利用GeXP多重基因表达遗传分析系统,建立一种多重逆转录-聚合酶链反应(mRT-PCR)方法,同时检测与病毒性脑炎相关的乙型脑炎病毒(Japanese encephalitis virus,JEV)等8种虫媒病毒。优化多重反应体系及反应条件,分别以病毒分离培养物和阳性标本来验证多重反应体系的特异性,以克隆质粒体外转录的RNA梯度稀释液来检测多重检测体系的灵敏度。结果表明,优化后的多重检测体系,可扩增出各病毒对应的特异片段,并可在102拷贝/μL水平同时并特异地检测出8种(共13个特异片段)脑炎相关病毒RNA。该方法具有高通量、灵敏度高、特异性强且快速等优点,对病毒性脑炎的分子诊断及流行病学调查具有重要意义。Multiplex reverse transcription-polymerase chain reaction（mRT-PCR） is currently available in virus detection and defined as the simultaneous amplification of two or more DNA/RNA targets in a single reaction vessel.In this study,we attempted to modify the conventional mRT-PCR technique on a basis of GenomeLab Genetic Analysis System（GeXP）.Initially,we optimized the analytical validation of the GeXP analyzer and its design of workflow and simultaneously detected eight arboviruses that related to epidemic encephalitis by verifying the specificity of mRT-PCR with Japanese encephalitis virus（JEV） cell cultures and positive strains identified previously and determining the sensitivity with in vitro-transcribed RNA of serial dilutions.The GeXP system after optimization could amplify the specific fragments related to the viruses and exposed specifically a total of 13 target genes out of eight types of arboviruses at the level of 102copies/μL,and the findings suggest that the novel protocol we developed can be high-throughput and highly specific and sensitive as well as quickness in screening of the encephalitis viruses,and is promising in detection of encephalitis-associated viruses for molecular epidemiological studies.

关 键 词：GeXP系统 病毒性脑炎 多重RT-PCR 

分 类 号：R373.3[医药卫生—病原生物学]