可变串联重复序列分析在辽宁省结核分枝杆菌基因分型中的应用

Genotyping of Mycobacterium tuberculosis in Liaoning with variable number of tandem repeats method

目的了解辽宁省81例结核分枝杆菌多位点数目可变串联重复序列分析(variable number of tandem repeats,VNTR)的基因多态性及近期传播情况。方法对2018年辽宁省内送至辽宁省疾病预防控制中心实验室的菌株进行传代后采用对硝基苯甲酸(p-nitrobenzoic acid,PNB)培养基进行菌群鉴定,以区分结核分枝杆菌复合体和非结核分枝杆菌。采用固体比例法利用罗氏培养基对4种一线抗结核药物进行药敏实验。以PCR为基础对标准24位点进行检测分析,利用RD105基因缺失法鉴定北京基因型。利用Microsoft Excel软件进行多态性分析,利用https://www.miru-vntrplus.org/MIRU/index网站得到VNTR基因分型结果,同时进行位点多态性和成簇率的计算。采用SPSS 23.0软件进行数据的统计描述,对计数资料或各年度耐药情况进行χ~2检验;当理论频数<5时,采用Fisher确切概率法计算双侧P值,检验水准α=0.05。结果经分析81株菌株可分为3个基因群(Ⅰ、Ⅱ、Ⅲ),菌株数分别为1(1.23%)、4(4.94%)和76(93.83%),其中最大的基因群为Ⅲ群。成簇率为4.94%(4/81)。近期感染率最小估计值为2.47%(2/81)。24个VNTR位点中有5个位点的多态性较好,3个位点的多态性尚可,16个位点的多态性较差。结论初步证实辽宁省耐药结核分枝杆菌菌株存在明显的基因多态性,其主要流行型为北京基因型(Ⅲ群)。

Objective To explore the genotype distribution and transmission patterns of 81 Mycobacterium tuberculosis(MTB)clinical isolates from Liaoning province with the variable number tandem repeats(VNTR).MethodsThe 81 strains were isolated from each city of the province in 2018,the p-nitrobenzoic acid(PNB)was used to identify the flora,so as to distinguish Mycobacterium tuberculosis complex from non-tuberculous mycobacteria.The solid ratio method was used to conduct drug susceptibility tests on four first-line anti-tuberculosis drugs using Roche’s medium.Based on PCR,the standard24 sites were detected and analyzed,and the Beijing genotype was identified by the RD105 gene deletion method.Microsoft Excel software was used for polymorphism analysis,and website(https://www.miru-vntrplus.org/MIRU/index)was used to get the VNTR genotyping results,the locus polymorphism and clustering rate at the same time was calculated.SPSS 23.0 software was used for statistical description of data,andχ~2 test carried out on count data or drug resistance in each year;when the theoretical frequency is less than 5,the Fisher exact probability method is used to calculate the two-sided P value,and the test level isα=0.05.ResultsThe clustering analysis of genotype showed that 81 strains were categorized into 3 gene clusters(genogroupⅠ,ⅡandⅢ),in which 1.23%(1/81)was belong to genogroupⅠ,4.94%(4/81)was genogroupⅡ,and 93.83%(76/81),one of the largest group of genes forⅢgroup.The clustering rate was 4.94%(4/81)and the least estimated proportion of the recent infection rate was 2.47%(2/81).The polymorphism of 5 in 24 VNTR loci was better,3 loci was acceptable,the remaining 16 loci was less polymorphic.ConclusionThe drug resistant Mycobacterium tuberculosis shows obvious gene polymorphisms in Liaoning province and Beijing genotype is the predominant prevalent strain(Ⅲgroup).