登革病毒Taqman双重荧光PCR分型研究  

Typing of dengue virus Taqman with double real-time PCR

作　　者：董瑞玲[1] 甄胜西[1] 孙杰[1] 李微[1] 王佃鹏[1] 徐媛[1] 朱玉兰[1] 

机构地区：[1]深圳国际旅行卫生保健中心,广东深圳518033

出　　处：《中国热带医学》2011年第12期1438-1439,1459,共3页China Tropical Medicine

基　　金：深圳出入境检验检疫局科研计划项目(No.SZ2008011)

摘　　要：目的建立鉴定登革病毒型别的双重实时Taqman PCR反应体系,以准确快速鉴定登革病毒型别。方法根据GenBank上已发表的登革病毒四个型别的全基因序列,进行对比分析,分别设计登革病毒的四个型别引物和探针,登革I、III型探针用FAM-TAMARA标记,登革II、IV型探针用JOE-TAMARA标记。经过条件优化后,建立检测登革病毒I/II型和III/IV型的两套双重实时荧光RT-PCR方法,扩增四型登革病毒RNA、登革病毒阴性样本和登革病毒RNA稀释样本,检测方法的特异性、重复性和检测限性。结果通过设计筛选序列和优化反应条件,建立登革病毒I、II型和登革病毒III、IV型的双重荧光PCR反应体系,通过试验证明,所建立的方法具有良好的特异性、重复性和检测限性,能准确快速地对登革病毒进行分型。结论建立了一种快速双重荧光PCR方法能同时对登革病毒进行分型和鉴定。Objective To establishing a multiplex real-time Taqman PCR method to quickly and correctly identify dengue virus type.Methods According to the gene sequences of the four dengue types from the GenBank,four series of dengue virus type-specified primers and probes were designed.Dengue virus I and III＇s probes were labelled with FAM-TAMARA,while dengue virus II and IV＇s probes were labelled with JOE-TAMARA.The reaction condition was optimized.Two multiplex real-time PCR methods were established to identify dengue virus I and II and dengue virus III and IV accordingly.Four type dengue virus RNA,dengue virus negative samples,dengue virus RNA diluted samples were tested to identify the specificity,reproducibility and sensitivity of the method.Results The dengue virus I and II（dengue virus III and IV）multiplex typing real-time PCR method could quicky identify dengue virus type witjh good specificity,reproducibility and sensitivity.Conclusion A multiplex real-time Taqman PCR method were established that can quickly identify dengue virus types.

关 键 词：登革病毒 基因分型 双重实时PCR TAQMAN探针 

分 类 号：R373.33[医药卫生—病原生物学]