分枝杆菌简单序列重复区间分型方法的研究

Optimization of inter-simple sequence repeat （ISSR） analysis of Mycobacterium

目的构建分枝杆菌简单序列重复区间（inter-simple sequence repeat，ISSR）分型系统，为结核分枝杆菌的基因分型、菌种鉴定以及流行病学的研究提供新的研究工具和研究思路。方法根据结核分枝杆菌基因组中简单重复序列（SSR）的丰度和频率，设计用于分枝杆菌基因分型的ISSR引物，以分枝杆菌属的DNA为材料，分析DNA浓度、Mg^2＋浓度、dNTP浓度、Taq酶的含量以及退火温度对ISSR-PCR扩增结果的影响，构建分枝杆菌的ISSR分型体系，并通过该体系筛选获得扩增结果稳定、多态性强的引物。结果经过优化实验建立了分枝杆菌ISSR-PCR最佳反应体系，20μl的PCR反应液含有的组分和终浓度分别为：10ng模板DNA，1U Taq酶，0．5μmol／L引物，1．5mmol／L MgCl2，0．2mmol／L dNTPs。利用该反应体系从10个ISSR引物中，筛选出了3个稳定性高、重复性好的引物，对分枝杆菌属28个菌种的标准菌株及52株结核分枝杆菌临床分离株进行了ISSR遗传多样性分析。结果显示，28个分枝杆菌菌种，除了结核和牛分枝杆菌之间具有较高的相似性，两两之间存在着高度的遗传多样性；52株结核分枝杆菌菌株在用ISSR进行分型时，大部分菌株表现出了较高的遗传相似性。结论建立了分枝杆菌属的ISSR分型体系。

Objective To establish inter-simple sequence repeat （ISSR） molecular markers in Mycobacterium. Methods Based on SSR of genome in Mycobacterium, ISSR primers were designed. The factors which affected the ISSR analysis of Mycobacterium, such as annealing temperature and concentrations of template DNA, Taq DNA polymerase, Mg^2＋ , dNTP were examined to establish optimal ISSR-PCR reaction system. And primers which can be used to analyze genetic variation in Mycobacterium were selected. Results The optimal ISSR-PCR reaction system （20μl） in experiments was as the following： 1×Taq buffer, 10 ng template DNA, 1U Taq DNA polymerase,0.5 μmol/L primer, 1.5 mmol/L MgCl2 ,0.2 mmol/L dNTPs. Of the 10 primers screened, 3 ISSR primers produced highly reproducible and polymorphic bands. The results of ISSR analysis in Mycobacterium showed that 28 reference strains from Mycobacterium had high level of genetic diversity except between Mycobacterium tuberculosis and Mycobacterium bovis, while 52 strains from M. tuberculosis had high genetic similarities. Conclusion ISSR molecular markers were successfully established in Mycobacterium.