结核分枝杆菌多位点可变数目串联重复序列分型方法标准化操作程序的建立  

Establishment of the standard operation program on multiple loci variable numbers tandem repeats analysis typing on Mycobacterium tuberculosis

作　　者：吕冰[1] Christine Pourcel 刘敬华[1] 董海燕[1] 张媛媛[1] 蒋毅[1] 刘志广[1] 赵秀芹[1] 万康林[1] 

机构地区：[1]中国疾病预防控制中心传染病预防控制所传染病预防控制国家重点实验室,北京102206 [2]Polymorphism and Minisatellites(GPMS),Institute of Genetics and Microbiology(IGM),France

出　　处：《中华流行病学杂志》2008年第9期919-924,共6页Chinese Journal of Epidemiology

基　　金：国家自然科学基金资助项目（30771853）

摘　　要：目的建立结核分枝杆菌多位点可变数目串联重复序列分析（MLVA）的标准化方法，评价该方法的分型能力、应用价值。方法选取15个位点，采用核酸提取、PCR和琼脂糖凝胶电泳等技术，结合BioNumerics（Version5．0）软件，对中国54株结核分枝杆菌临床分离株进行分型。结果确定标准化的MLVA方法，包括细菌分离培养、核酸提取、PCR、琼脂糖凝胶电泳等实验步骤及标化参数的相关数据分析软件的使用。VNTR位点确定为ETRA、ETRB、ETRC、ETRD、ETRE、MIRU10、MIRU16、MIRU23、MIRU26、MIRU27、MIRU39、MIRU40、Mtub21、Mtub30和Mtub39。结论建立MLVA标准化技术方案，该方法操作简便，分型鉴定能力及实验室间结果可比性强，便于网络化，有利于结核病流行中追溯传染源，分析流行趋势。Objective To establish and evaluate the standardized protocol of multiple loci variable numbers tandem repeats （VNTR） analysis （MLVA） for genotyping Mycobacterium tuberculosis （M. tuberculosis ）. Methods 15 VNTR loci were chosen for genotyping 54 Chinese M. tuberculosis strains by PCR-electrophoresis-based VNTR analysis and the results were analyzed by software BioNumerics （Version 5.0 ）. Results MLVA method was successfully established and standardized, including the standard protocol for bacterial culture, DNA isolation, PCR and agarose gel electrophoresis and the software analysis. 15 VNTR loci were confirmed, including ETRA, ETRB, ETRC, ETRD, ETRE, MIRU10, MIRU16, MIRU23, MIRU26, MIRU27, MIRU39, MIRU40, Mtub21, Mtub30 and Mtub39, to be suitable for MLVA analysis of M. tuberculosis. Conclusion The standardized MLVA method has been established successfully. This method is simple and has powerful capacity for genotyping and strain differentiation, can be used for the network surveillance on pathogens of M. tuberculosis, and the data are comparable between laboratories. It is valuable for tracing the source and studying the trend of prevalence during investigation of M. tuberculosis infections.

关 键 词：结核分枝杆菌 多位点可变数目串联重复序列分析 标准化操作程序 

分 类 号：R378[医药卫生—病原生物学]