【关键词】 结核分支杆菌；rPOB基因；KatG基因；rpsL基因；SSCP；药物耐受性
Studies of Mycobacterium tuberculosis multi-drug resistant gene
【Abstract】 Objective To evaluat the importance of rPOB、KatG and rpsL gene mutation detection in multi-drug resistant clinical isolates of Mycobacterium tuberculosis. Methods rPOB、KatG and rpsL gene mutation of 54multi-drug resistant clinical isolates，which is INH RFP and Sm resistance and 54 drug susceptible strains were analyzed using polymerase chain reaction-single strand conformation (PCR-SSCP).Results SSCP pattern of H37RV strain as control，SSCP pattern of rPOB，rpsL PCR amplification from drug susceptible strains were all the same as control. The specificity was 100%. However，the different SSCP pattern of KatG gene were found in 2 susceptible strains. The specificity was 96.3%. Gene deletion of rPOB、KatG and rpsL were not found in all clinical isolates tested. SSCP patterns of rPOB gene were different from control in 49 drug resistant clinical isolates. SSCP patterns of KatG gene were different from control in 32drug resistant clinical isolates，SSCP pattern of rpsL gene were different from control in 40 drug resistant clinical isolates. The sensitivity were 90.1% (rPOB)，59.3% (KatG) and rpsl (74.1%)，respectively. Conclusion The major mechanism of RFP，INH，SM resistant drug was rPOB、KatG and rpsL gene mutation，respectively PCR-SSCP may be one of the way which applied to drug resistant detection of Mycobacterium tuberculosis.
【Key words】 Mycobacterium tuberculosis；rPOB gene；KatG gene；rpsL gene；SSCP；drug tolerance
我国是结核病疫情最严重的国家之一，其主要原因是耐药（DR-TB）和耐多药（MDR-TB）结核病的广泛分布和迅速传播。由于单药化疗和不规则化疗，其耐药情况越来越严重。近年来随着分子生物学的研究进展人们对抗结核分枝杆菌的药理作用位点和结核分枝杆菌耐药机制，尤其是耐异烟肼、利福平和链霉素。目前，结核分支杆菌耐药性测定仍多采用绝对浓度法、比例法等经典方法。但由于结核分支杆菌生长缓慢，而耐药结核分支杆菌生长更为缓慢，其耐药性测定需6～8周甚至更长，不能及时指导临床用药。新进开展的Bactec法药敏检测因其价格昂贵等诸多不便限制了其广泛使用。据报道［1］INH耐药与过氧化氢酶-过氧化物酶的编码基因（KatG）有关、而某些结核分支杆菌耐SM的产生是由于其核糖体蛋白S12编码基因（rpsL）和16SrRNA编码基因（rrs）突变所致［2］，同时也证实了结核分支杆菌耐利福平与RNA聚合酶的β亚基的编码基因rPOB的突变有关［3］。笔者采PCR-SSCP银染法对54例（同时耐INH、RFP、SM）和45例（药物敏感）肺结核病人痰标本临床分离株同时进行KatG基因、rPOB基因和rpsL基因突变分析。
1 材料与方法
1.1 菌株来源 H37RV标准株来源于中国药品生物制品检定所，45例敏感株和54例同时耐INH、RFP、SM的临床分离株来自珠海市人民医院、沈阳市胸科医院等。按结核病诊断细菌学规程进行菌种鉴定及药敏试验证实为结核分支杆菌，耐药株则同时对异烟肼、利福平、链霉素耐药。药物的最高浓度为异烟肼10mg/L、利福平250mg/L、链霉素100mg/L。
1.2 方法
1.2.1 细菌DNA制备 临床分离株DNA用常规酚/氯仿提取，并将纯化后的提取物置于－20℃保存备用。
1.2.2 PCR扩增 采用25μl反应体系，4xdNTPs终浓度为0.2mmol/L，引物终浓度为0.2μmol/L，置DNA扩增仪扩增，扩增产物经2%琼脂糖凝胶电泳。紫外检测rPOB出现258bp条带、KatG出现282bp条带、rpsL出现267bp条带即为扩增阳性。
1.2.3 SSCP银染 PCR扩增阳性的标本进行SSCP检测，扩增产物加等量甲酰胺变性液95℃变性10min，立即冰浴5min，在8%非变性聚丙烯酰胺凝胶中电泳，条件为6℃、100V电压，电泳约2～3h，电泳结束后，取凝胶板行硝酸银染色，观察结果并照相。
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