基于DTM-PCR、MIRU-VNTR和耐药基因测序的武汉市耐多药结核分枝杆菌的分子分型

Molecular typing of multidrug-resistant mycobacterium tuberculosis based on DTM-PCR,MIRU-VNTR,and sequencing of drug-resistance-associated genes in Wuhan

目的描述武汉市耐多药结核(multidrug-resistant tuberculosis,MDR-TB)分枝杆菌基因型分布及成簇特征。方法采用目标缺失多重PCR(Deletion-Targeted Multiplex,DTM-PCR)、24位点MIRU-VNTR(mycobacterial interspersed repetitive unit-variable number tandem repeat typing)以及耐药基因测序方法,对武汉市149株耐多药结核分枝杆菌进行分子分型,计算Hunter-Gaston分辨指数(Hunter-Gaston discriminatory index,HGDI),成簇率以及近期感染率最小估计值。结果全部MIRU位点的累积HGDI为0.9944。QUB11b位点的HGDI最高(0.6738)。联合采用DTM-PCR以及MIRU-VNTR,共鉴定出111个独立基因型和11个成簇基因型(38株),成簇率为25.50%,近期感染率最小估计为18.12%。经过耐药基因测序进一步分型,各簇内共有17(44.74%)个菌株有相同的突变位点,其余21个菌株突变位点不相同。结论联合采用DTM-PCR、24位点MIRU-VNTR分型和耐药基因测序,其基因分型分辨率较高,适合于武汉地区耐多药结核分枝杆菌的基因分型。武汉市MDR-TB的发生大多由于内源性复燃引起,但仍有一定的近期传播率。

Objective To determine the genotype and clustering characteristics of multidrug-resistant(MDR)mycobacterium tuberculosis in Wuhan City.Methods A total of 149 mycobacterium tuberculosis strains from MDR tuberculosis(MDR-TB)patients were genotyped by DTM-PCR,mycobacterial interspersed repetitive unit–variable number tandem repeat typing(MIRU-VNTR)and sequencing of drug-resistant associated genes.The Hunter-Gaston discriminatory index(HGDI),clustering rate and recent minimum estimated infection rate were calculated.Results The cumulative HGDI of all MIRU loci was 0.9944.Gene locus with the highest HGDI(0.6738)was QUB11b.Combing MIRU-VNTR and DTM-PCR,111 unique genotypes and 11 clustered genotypes(38 strains)from 149 isolates were identified.The clustering rate was 25.50%,and the recent minimum infection rate was estimated to be 18.12%.After further genotyping by sequencing of drug-resistant associated genes,the results showed that 17(44.74%)strains in each cluster shared the same mutation sites,while the other strains had different mutation sites.Conclusion The combination of DTM-PCR,24 locus MIRU-VNTR typing and drug resistance gene sequencing demonstrates a relatively high discriminatory power,which is suitable for the genotyping of multi drug resistant mycobacterium tuberculosis in Wuhan.The epidemic of MDR-TB in Wuhan is mainly caused by reactivation,and the recent transmission rate is not high.