西尼罗病毒一步实时RT-PCR检测准确度评价  

Evaluation of the accuracy of one step Real-time RT-PCR on detecting West Nile virus

作　　者：孙后超[1,2] 徐鸣明[1,2] 邓永强[3] 何丰[1,2] 冯裕星[1,2] 胡子成[1,2] 秦鄂德[3] 谢鹏[1,2] 秦成峰[3] 

机构地区：[1]重庆医科大学附属第一医院神经内科,重庆400016 [2]重庆医科大学神经科学研究中心 [3]军事医学科学院微生物流行病研究所,病原微生物生物安全国家重点实验室,北京100071

出　　处：《解放军医学杂志》2010年第6期683-686,共4页Medical Journal of Chinese People's Liberation Army

基　　金："十一五"国家高科技发展(863)计划(2006AA02Z196)

摘　　要：目的了解已建立的一步实时RT-PCR对西尼罗病毒感染细胞和组织的灵敏度及其对国内常见的黄病毒属病毒的特异性,为西尼罗病毒感染的实验室检测和流行病学调查奠定基础。方法使用VectorNTISuite8软件比较探针与西尼罗病毒不同系毒株之间的同源性。采用西尼罗病毒感染的Vero细胞培养上清和BALB/c小鼠脑组织样本评价一步实时RT-PCR法的灵敏度,同时用国内常见的黄病毒属病毒评价其特异性。结果此一步实时RT-PCR法可用于西尼罗病毒1系多数毒株的检测,灵敏度高,对西尼罗病毒的最低检出浓度为5.75×10-2pfu/ml;与黄病毒属乙型脑炎、登革热、蜱传脑炎以及甲病毒属东部马脑炎等虫媒病毒均未出现交叉反应。结论西尼罗病毒一步实时RT-PCR法具有灵敏度高、特异性强的特点,适用于西尼罗病毒感染细胞和组织的早期检测及流行病学调查。Objective The accuracy including sensitivity and specificity of the one step real-time reverse transcriptase PCR that have already been established for detecting West Nile virus was systemically evaluated with different genera of Flavivirus virus in the present study,rendering it more reliable and accurate in application of this method to detect West Nile virus not only in laboratory diagnosis but also epidemiological investigation. Methods The probe for the one step real-time reverse transcriptase PCR was designed from the alignment of the sequences of West Nile virus lineage 1 strains and lineage 2 strains using Vector NTI Suite software Ver. 8.0. The sensitivity of this method was evaluated by the Vero cell and BALB/c mouse brain tissue infected with West Nile virus,while the specificity was determined with other different Flavivirus sp. of the family Flaviviridae,such as Japanese encephalitis virus,Dengue virus,tick borne encephalitis virus,and also eastern equine encephalomyelitis virus belonging to the genus Alphavirus of the family Togaviridae. Results The one step real-time RT-PCR method could be utilized for detecting most of West Nile virus lineage 1 strains with high sensitivity,and the lowest detectable concentration of West Nile virus was calculated as 5.75×10-2pfu/ml. No cross reactions were observed with Japanese encephalitis virus,Dengue virus,tick borne encephalitis virus and eastern equine encephalomyelitis virus. Conclusion The method is characterized with high sensitivity and specificity,and is accurate enough for the application of quantitative detection of West Nile Virus RNA for epidemiological survey and laboratory studies.

关 键 词：西尼罗病毒 逆转录聚合酶链反应 分子探针技术 敏感性与特异性 

分 类 号：R446.52[医药卫生—诊断学]