45个可变数目串联重复序列位点用于中国结核分枝杆菌基因型鉴定的分辨力评价

摘要

Objective To evaluate the discriminatory efficiency of multiple loci of variable numbers of tandem repeats (VNTR) in Mycobacterium tuberculosis genome.Genotyping and identification on Chinese M.tuberculosis clinical strains were used to locate a series of high discriminated loci,so as to provide the basis for creating a standardized multiple loci VNTR analysis (MLVA) to distribute fast typing and identification on Chinese M.tuberculosis.Methods VNTR loci which were chosen from the website http://minisatellites.u-psud.fr/ and referenced to the genome sequence of M.tuberculosis standard strain H37Rv were tested in Chinese M.tuberculosis clinical strains and H37Rv by means of PCR.The primers were designed by DNAStar software.The repeat member of VNTR unit was estimated by the result of PCR.The discrimination power of single locus or multiple loci was confirmed by the Hunter-Gaston index.Results 45 VNTR loci were tested in 135 Chinese M.tuberculosis clinical strains and H37Rv.The discrimination power of these loci appeared different from each other,with the biggest Hunter-Gaston index as 0.814 (0.797-0.830),the smallest one as 0.015 (0.001-0.028),and there were 13 loci with which the Hunter-Gaston index was bigger than 0.5.Results showed that the discrimination power was increasing by different loci that associated with each other.The more loci that were combined,the bigger the Hunter-Gaston index was,For example,the Hunter-Gaston index of Qub11-b associated with Qub 18 was 0.936,by which 136 strains could be divided into 44 groups.With the combination of 9 loci including Qub11-b,Qub18,Mtub21,Rv2372,MIRU26,Qub26,Qub4156c,Qub11-a and Qub15,the HunterGaston index could have reached 1 and by which the 136 strains could be divided into 136 groups,also showing that the biggest discrimination power to strain identification,viz.strain level genotype were reached.Conclusion The discrimination power of different locus was different.The discrimination power of multiple loci was much more satisfied than that of single locus.It was satisfied the combine discrimination power of 9 loci including Qubll-b,Qubl8,Mtub21,Rv2372,MIRU26,Qub26,Qub4156c,Qub11-a and Qubl5,by which the qualified typing method could gain to facilitate research on molecular epidemiology with the Hunter-Gaston index analysis.%目的 评价结核分枝杆菌基因组中串联重复序列(VNTR)位点在中国结核分枝杆菌临床分离株的分型鉴定的分辨力.方法 参考http://minisatellites.u_psud.fr/网站记载的位点选取其中VNTR位点,参照H37Rv结核分枝杆菌标准株全基因序列,利用DNAStar软件设计引物,采用PCR分别对中国结核分枝杆菌临床分离株和H37Rv标准株的VNTR位点进行检测,根据PCR凝胶电泳图片中各菌株相应位点扩增产物的大小,确定重复单元的重复次数.计算Hunter-Gaston指数来分析各位点的分辨能力,及各位点合并分辨力.结果 共检测135株中国结核分枝杆菌临床分离株和H37Rv标准株的45个VNTR位点.结果显示45个VNTR位点对136株菌的分辨力各不相同,Hunter-Gaston指数最大者为0.814(0.797～0.830),最小者为0.015(0.001～0.028),＞0.5的有13个位点.位点的合并分辨力分析显示,随着位点数量增加,分辨力增强,如Qub11-b和Qub18两个位点合并分析,Hunter-Gaston指数为0.936,分组数为44组;Qub11-b、Qub18、Mtub21、Rv2372、MIRU26、Qub26、Qub4156c、Qub11-a和Qub15等9个位点合并分析,Hunter-Gaston指数已经达到1,组数为136组,表示已达到最大分辨力,即株水平分型.结论 不同VNTR位点具有不同的分辨力.其分型分辨力,多位点联合明显好于单位点.Qub11-b等9个位点的合并分型能力较好,这些位点有利于结核分枝杆菌的分型研究.