贵州省部分地区结核分枝杆菌MLVA基因分型研究

Genotyping of Mycobacterium tuberculosis in Guizhou Province using MLVA method

目的应用多位点可变串联重复序列分析技术(MLVA)对贵州省结核分枝杆菌进行基因分型分析,为贵州省结核病防控提供科学依据。方法选取贵州省150株临床分离株结核分枝杆菌,采用水煮法提取基因组DNA,PCR扩增15个VNTR位点,统计菌株各VNTR位点的重复数目,通过遗传差异值(h)及Hunter-Gaston指数对VNTR位点进行遗传多态性及分辨力评价,采用BioNumerics 5.0软件对各菌株进行聚类关系和最小间距图(minimum spanning tree,MST)分析。结果 PCR检测结核菌株VNTR各位点呈明显多态性,以Mtub21和MIRU26多态性尤为显著,以h值分别为0.559和0.505;MIRU10和ETRB显示较低基因多态性,h值分别为0.052和0.090。聚类分析显示,150株菌株分为Ⅰ、Ⅱ、Ⅲ、Ⅳ4个基因群,其中Ⅰ群占10.67%(16/150),Ⅱ群占30.67%(46/150),Ⅲ群占40.00%(60/150),Ⅳ群占18.67%(28/150)。4个基因群呈现明显地域分布,毕节市以Ⅰ、Ⅱ群为主要流行菌株,安顺市以Ⅲ群菌株为主要流行菌株,遵义市以Ⅳ群菌株为主要流行菌株。MST分析显示,150株菌株形成3个克隆复合体(clonal complexes,CCs)及若干个独立分支(singleton),遵义、安顺、毕节分离株分别分布于不同的克隆复合体CCs。结论贵州省结核分枝杆菌存在明显的基因多态性和地域分布特性,以Ⅲ群和Ⅱ群菌株为主要流行菌株,应加强对上述两群菌株的监控。

Objective To investigate the genotype distribution of Mycobacterium tuberculosis strains from Guizhou Province with multiple locus variable number tandem repeats analysis(MLVA) in order to provide strategies for the prevention and control of tuberculosis in Guizhou Province. Methods 150 M. tuberculosis clinical isolates were randomly selected from Guizhou Province, and then their genome DNA was extracted by boiling-water method. The repeat numbers of 15 VNTR loci in the DNA samples was analyzed by PCR technique, respectively. The genetic polymorphism and discrimination was evaluated by allelic diversity(h) and Hunter-Gaston index(HGI). The clustering of genotype and minimum spaning tree(MST) was analyzed with the software BioNumerics(Version5.0). Results there were obvious polymorphisms of VNTRs in 150 strains, Mtub21 and MIRU26 were highly discriminative(h=0.559 and 0.505, respectively). Contrary, MIRU10 and ETRB were poorly discriminative(h=0.052 and 0.090, respectively). The clustering analyses of genotype showed that 150 strains were categorized into 4 gene clusters(genogroup Ⅰ, Ⅱ, Ⅲ and Ⅳ), in which 10.67%(16/150) was belong to genogroup Ⅰ, 30.67%(46/150) was genogroup Ⅱ, 40.00%(60/150) was genogroup Ⅲ, and 18.67%(28/150) was belong to genogroup Ⅳ. Interestingly, 4 gene clusters showed obvious geographical distribution, the genogroup Ⅰ and Ⅱ were prevalenting in Bijie City, the dominating gene cluster was genogroup Ⅲ in Anshun City, the main prevailing strain in Zunyi City was genogroup Ⅳ. The MST results showed that 150 strains were categorized into 3 clonal complexes(CCs) and several separate singletons. The strains from Zunyi、Anshun and Bijie belongs to difference clonal complex, respectlively. ConclusionM. tuberculosis clinical strains from Guizhou Province had obvious VNTR polymorphisms. The genogroup Ⅱ and Ⅲ strains were predominantly prevalent in Guizhou Province, the surveillance on these two types of strains should be strengthened.