TaqMan-MGB探针实时荧光PCR检测结核分枝杆菌北京基因型的研究

Identification of Beijing genotype of Mycobacterium tuberculosis by real-time PCR with TaqMan-MGB probe

目的探讨TaqMan-MGB探针实时荧光PCR快速检测结核分枝杆菌北京基因型菌株的效果。方法对杭州地区136株结核分枝杆菌临床分离株水煮法提取DNA,分别用TaqMan-MGB探针实时荧光PCR与间隔区寡核苷酸(Spoligotyping)基因分型方法进行北京基因型菌株鉴定,比较2种方法的一致性,同时评价TaqMan-MGB探针实时荧光PCR的最低检测限。结果以Spoligotyping基因分型方法为标准,136株结核分枝杆菌中,检测到北京基因型结核分枝杆菌105株,占77.21%。TaqMan-MGB探针实时PCR方法与Spoligotyping基因分型方法鉴定北京基因型菌株结果完全一致。TaqMan-MGB探针实时荧光PCR对北京基因型最低检出限为0.488 pg/μl。结论与Spoligotyping基因分型方法相比,TaqMan-MGB探针实时PCR方法检测北京基因型操作简单,耗时短,灵敏度高,可以快速地区分北京基因型与非北京基因型结核分枝杆菌。

Objective To know about the application effect of TaqMan-MGB probe based real-time PCR for the rapid identification of Beijing genotype Mycobacterium tuberculosis.Methods A simple method of DNA extraction using boiling water-bath was performed for 136 Mycobacterium tuberculosis isolates from Hangzhou area.All Beijing genotype strains were identified by TaqMan-MGB probe based real-time PCR and Spoligotyping,respectively.The consistency of the two methods was compared,and the minimum detection limit of TaqMan-MGB probe on real-time fluorescence PCR was evaluated.Results According to the Spoligotyping results as a standard,among all of the 136 MTB strains,77.21%（105 isolates） were determined as Beijing genotype.The TaqMan-MGB probe based real-time PCR could identify all of the Beijing strains with 100% consistency.The minimum detection limit of the TaqMan-MGB probe based on real-time PCR was 0.488 pg/μl.Conclusion Compared with Spoligotyping,the TaqMan-MGB probe based real-time PCR could rapidly differentiate Beijing strains or non-Beijing strains of Mycobacterium tuberculosis,and it is simple,accurate and sensitive.