IS6110限制性片段多态性分析标准方法的建立及其在结核分枝杆菌分子分型中的应用

Establishment and application of a standard IS6110-RFLP method in the study of molecular genotyping analysts on Myobacterium tuberculosis

目的建立IS6110限制性片段多态性分析（IS6110一RFLP）标准方法并评价该方法的分型能力。方法采用核酸提取、PCR、限制性内切酶分析、Southern杂交、琼脂糖凝胶电泳等技术，结合Gel—Proanalyzer3．1和BioNumerics（Version5．0）软件，对78株结核分枝杆菌插入序列IS6110-RFLP进行分析。结果确定标准化的IS6110-RFLP技术，包括核酸提取、PCR、限制性内切酶分析、Southern杂交、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；采用该技术，将78株结核分枝杆菌分为75个不同的基因型，分别归属于11个基因簇，其中有52株归属于同一个基因簇，占菌株总数的66．7％（52／78）。结论建立标准化的IS6110-RFLP技术方案，该方法具有很强的基因分型和株水平鉴定能力，可用于结核病的病原学监测。

Objective To develop a standardized IS6110-restriction fragment length polymorphism （RFLP） method, used for evaluating the capacity of genotyping. Methods IS6110-RFLP of 78 Mycobacterium （M.） tuberculosis strains were studied by bio-molecular techniques including DNA isolation, PCR, restriction endonuclease enzyme analysis, southern blotting, agarose gel electrophoresis, together with data analysis by software Gel-Pro analyzer 3.1 and BioNumerics （Version 5.0）. Results IS6110-RFLP method was established and standardized successfully, including DNA isolation, PCR, restriction endonuclease enzyme analysis, southern blotting, agarose gel dectrophoresis and usage of the analysis software with standard parameters. By this method,78 M. tuberculosis isolates were classified into 75 genotypes which belonged to 11 different clusters. Of all the isolates, 66.7% （52/78） belonged to a main cluster. Conclusion Standard IS6110-RFLP method was established successfully. This method had powerful capacity for genotyping and strain level identification and could be used for the surveillance on pathogens of M. tuberculosis in China.