乙型脑炎病毒TaqMan逆转录聚合酶链反应检测方法的建立  

Establishment of a TaqMan reverse transcription polymerase chain reaction for detection of Japanese encephalitis virus

作　　者：黄福新[1] 高世同[1] 张仁利[1] 钟木生 耿艺介[1] 黄达娜[1] 

机构地区：[1]深圳市疾病预防控制中心,广东深圳518020 [2]深圳市布吉坂田预防保健所,广东深圳518129

出　　处：《中国热带医学》2008年第9期1481-1482,共2页China Tropical Medicine

基　　金：深圳市科技计划资助项目(NO200603195)

摘　　要：目的采用TaqMan技术,研究建立实时荧光逆转录聚合酶链反应技术(RT-PCR)检测乙型脑炎病毒(Japanese encephalitis,JEV)的效果。方法根据JEV非结构蛋白基因NS3基因序列(GenBank序列号,U15763),结合生物学软件序列比对分析,设计1对特异性引物和TaqMan寡核苷酸探针,优化荧光RT-PCR反应条件后,以模板梯度稀释法及相关毒株对比扩增检验测定方法的敏感度和特异性。结果引物和探针最佳浓度均为0.20μM,复性温度为55℃。方法至少检测出1copy/μl病毒RNA,与流感、麻疹、腮腺炎、风疹、水痘等病毒均无交叉反应。从病毒核酸提取至完成检测仅需3hr左右。结论所建立的JEV病毒TaqMan荧光RT-PCR方法敏感特异,有助于JEV感染的实验室早期快速诊断。Objective To establish a specific and sensitive method for detecting Japanese encephalitis virus （JEV） nucle- ic acid with TaqMan reverse trascription polymerase chain reaction（RT-PCR）. Methods Based on NS3 nucleic sequnce of JEV from GenBank （NO. U15763 ）, a pair of primers and a TaqMan probe was designed for real -time RT- PCR. The reaction conditions were optimized using different concentration of primers and probe. Specificity and sensitivity of the TaqMan RT - PCR were also tested. Results The optimized concetration of the primers and probe was 0.20uM respectively. The RT - PCR was proved to be specific for detection JEV, with no cross - reactivity oberserved with influenza, measles, mumps, rubella and varicella viruses, which could detect as low as lcopy/ul of JEV RNA, The time was about 3hr for completing a detection process, Conclusion The established TaqMan real - time RT - PCR was specific and sensitive, which could be useful for early and rapid laboratory diagnosis of JE.

关 键 词：乙型脑炎病毒 荧光RT—PCR TAQMAN探针 检测 

分 类 号：R373.31[医药卫生—病原生物学]