结核分枝杆菌katG、inhA、ahpC等突变与异烟肼耐药关系的研究

目的：了解结核分枝杆菌katG、inhA、ahpC、fabG1、sodA及sodC基因突变的特征及其与耐异烟肼的关系。方法对127例活动性肺结核患者痰标本进行菌型鉴定及结核分枝杆菌药敏试验，提取结核分枝杆菌菌株DNA，应用PCR扩增katG、inhA及ahpC、fabG1、sodA及sodC基因片段，并进行DNA序列分析。结果结核分枝杆菌药物敏感试验显示127株结核分枝杆菌中，其中47株耐异烟肼，80株对异烟肼敏感，耐异烟肼率为37.01%。47株耐异烟肼中，29株存在katG和（或）inhA基因突变，其中22株（46.81%，22/47）存在katG基因单位点突变，3株（6.38%，3/47）存在inhA基因单位点突变，4株（8.51%，4/47）存在katG及inhA基因联合位点突变。22株katG基因单位点突变中，20株为AGC315ACC、AGC315AAC （42.55%，20/47）突变，2株（2.13%，1/47）分别为CTG378CCG（Leu378Pro）、ACG394ATG（Thr394Met）突变，该突变位点及突变形式尚未见文献报道。18株katG及inhA未突变结核分枝杆菌均未检测到ahpC、fabG1、sodA及sodC基因突变。结论结核分枝杆菌对异烟肼耐药主要与katG和inhA基因突变有关。耐异烟肼结核分枝杆菌临床分离株378和394新突变位点的发现为进一步研究耐药机制以及耐药结核病的快速检测提供了依据。

The relationship between mutations in katG,inhA, ahpC and other genes of Mycobacterium tuberculosis and isoniazid resistance

Objective To understand the relationship between characteristics of mutations in katG, inhA, ahpC, fabG1, sodA, and sodC genes of Mycobacterium tuberculosis and isoniazid （INH） resistance. Methods A total of 127 Mycobacterium tuberculosis clinical isolates from sputum of patients with active pulmonary tuberculosis were collected and used for mycobacterium species identification, drug susceptibility testing and extraction of genomic DNA. DNA fragments specific for katG, inhA, ahpC, fabG1, sodA and sodC genes were amplified by PCR and used for sequence analysis and comparison with the standard laboratory strains （H37RV） through the use of NCBI database. Results Out of 127 clinical isolates tested for INH susceptibility, 47 （37.01%） were resistant and 80 were susceptible to INH. Among 47 INH-resistant isolates, 29 had mutations in katG and/or inhA genes, 22 （46.81%, 22/47） had point mutation in katG gene, 3 （6.38%, 3/47） had mutation in the promoter region of inhA gene, and 4 （8.51%, 4/47） had point mutations in both katG and inhA genes. Among 22 isolates with mutation in katG gene, 20 （42.55%, 20/47） had mutation at codon 315 （AGC315ACC or AGC315AAC） and 2 （2.13%, 1/47） had mutation at codons 378 （CTG378CCG, Leu378Pro） and 394 （ACG394ACC, Thr394Met）. In 18 isolates without mutations in katG and inhA genes, no mutation was detected in ahpC, fabG1, sodA and sodC genes. Conclusion Results from this study further confirm that mutations in katG and inhA genes of Mycobacterium tuberculosis are associated with isoniazid resistance. Two new point mutations in the katG gene have been identified from the study, which will provide new information for further development of quick diagnostic methods for drug-resistant tuberculosis.