多重实时荧光定量RT-PCR法快速检测3种蚊媒病毒  

Rapid detection of three kinds of mosquito-borne virus by multiplex real-time fluorescence quantitative RT-PCR

作　　者：吴忠华[1] 罗鹏[1] 吕沁风[1] 郑伟[1] 

机构地区：[1]浙江国际旅行卫生保健中心,浙江杭州310003

出　　处：《中国卫生检验杂志》2015年第12期1975-1977,共3页Chinese Journal of Health Laboratory Technology

基　　金：质检总局科研课题(2012IK248);浙江省重点科技创新团队项目(2011R50021)

摘　　要：目的建立多重实时荧光定量RT-PCR快速检测方法,用于黄热病毒(YFV)、登革热病毒(DFV)、基孔肯雅热病毒(CHIKV)的同时检测和鉴别诊断。方法分别针对YFV 3'UTR基因、DFV 3'UTR C-M基因和CHIKV nsp2基因保守区设计特异性引物和Taq Man探针,建立并优化多重实时荧光定量RT-PCR反应体系,评价方法的特异性、敏感性和稳定性,并用临床标本进行验证。结果该方法可同时检测YFV、DFV和CHIKV,检测结果的特异性强,灵敏度达1×103copy/ml,结果的重复性很好,其变异系数分别为1.03%、0.90%和0.34%。临床确诊的11例DFV标本,5例CHIKV标本和10例黄热病疫苗标本核酸检测结果皆为阳性。结论研究建立的多重实时荧光定量RT-PCR方法可同时检测YFV、DFV和CHIKV,灵敏度高、特异性强、稳定性好,是一种可同时快速检测多种蚊媒病毒的新方法。Objective To establish a rapid multiple real- time fluorescence quantitative RT- PCR method for the simultaneous detection and confirming of the yellow fever virus（ YFV）,dengue virus（ DFV） and chikungunya virus（ CHIKV）. Methods Primers and Taq Man hydrolysis probes were designed according to the YFV 3＇UTR gene,DFV 3＇UTR C- M gene and CHIKV nsp2 gene conservative region. A multiple real- time fluorescence quantitative RT- PCR assay was established and optimized,as well as the specificity,sensitivity and the stability of the assay method,which were verified with the clinical samples. Results YFV,DFV and CHIKV could be simultaneously detected by the method with high specificity,sensitivity of 1 × 103 copy / ml and well-behaved repeatability. The coefficients of variation were 1. 03%,0. 90% and 0. 34%,respectively. Among the 11 cases of DFV samples,5 cases of CHIKV clinically diagnosed specimens and 10 yellow fever vaccine were detected nucleic acid positive.Conclusion The multiple real- time fluorescence quantitative RT- PCR method can simultaneously detect YFV,DFV and CHIKV with high sensitivity,intensity specificity,well- behaved reproducibility,which is a kind of new method used to fast detect multiple mosquito borne virus.

关 键 词：黄热病毒 登革热病毒 基孔肯雅热病毒 多重实时荧光定量逆转录聚合酶链式反应 检测 

分 类 号：R373.33[医药卫生—病原生物学]