31株结核分枝杆菌异烟肼耐药相关基因检测及序列分析

目的了解贵阳地区分离的结核分枝杆菌katG基因、inhA启动子和oxyR-aphc间隔区基因突变特征。方法对31株结核分枝杆菌临床分离株（异烟肼耐药株17株,异烟肼敏感株14株）的katG基因、inhA启动子和oxyRaphc间隔区进行DNA片段的PCR扩增,并进行测序分析。结果 17株异烟肼耐药菌株中有16株检出katG基因突变,其中70.5%（12/17）为315位密码子变异,且变异类型均为AGC→ACC,敏感株未发现315位点突变。11株敏感菌和4株耐药菌在463位密码子发生变异,变异类型均为CGG→TGG,变异率分别为78.6%（11/14）和23.5%（4/17）,差异无统计学意义。有12株耐药株的变异类型为katG基因双重位点突变,其中10株为katG315（AGC→ACC）和463（CGG→TGG）位变异,463（CGG→TGG）和299（GGC→AGC）变异及463（CGG→TGG）和419（GAC→CAC）位变异各1株。2株异烟肼耐药菌检出oxyR-aphC启动区G32A突变,其中1株为联合katG463和299位突变。结论贵阳株结核杆菌菌株异烟肼耐药基因突变具有多态性,主要的变异类型为katG315位点突变。

Isoniazid resistance and related gene mutation of 31 Mycobacterium tuberculosis clinical isolates in Guiyang area

Objective The purpose of this study is to analyze gene mutations related with isoniazid resistance of Mycobacterium tuberculosis in Guiyang area. Methods The katG,inhA,oxyR-aphC gene fragment of M.tuberculosis was amplified and sequenced.These strains included seventeen isoniazid resistant clinical isolates and fourteen isoniazid susceptible clinical isolates.Sequences of these strains were analyzed by CLC FreeWorkbench4.5and BioEdit. ResultsThe mutations of katGgene were found in 16 isoniazid resistant clinical isolates.70.5%（12/17）of drug-resistant strains showed katG315（AGC→ACC）mutation.katG463（CGG→TGG）was found in 11 isoniazid susceptible clinical isolates and 4isoniazid resistant clinical isolates.The mutation rate of this type of mutation in isoniazid sensitive strains and isoniazid resistant strains was 78.6%（11/14）and 23.5%（4/17）respectively.There were no significant difference of this rate between sensitive strains and resistant strains（χ^2=6.48,P〉0.05）.Double mutations at two separate loci of katG were detected in 12 isoniazid resistance isolates,including katG315（AGC→ACC）combined with 463（CGG→TGG）in 10 strains,463（CGG→TGG）combined with 299（GGC→AGC）in 1isolate and 463（CGG→TGG）combined with 419（GAC→CAC）in 1isolate.No mutation ininhA was found in this study.G32 Amutation of oxyR-aphCgene was found in 2isoniazid resistant strain. Conclusion The results showed the gene polymorphism of katG,inhA,oxyR-aphCin M.tuberculosis.katG315 is the dominant gene mutation related with isoniazid resistant M.tuberculosis in Guiyang.