结核分枝杆菌耐乙胺丁醇分子机制的研究

Studies on molecular mechanism of ethambutol resistance in Mycobacterium tuberculosis clinical isolates

目的 了解结核分枝杆菌耐乙胺丁醇分子机制 ,建立快速分子药敏试验方法。方法 通过聚合酶链反应 (PCR) -单链构象多态性 (SSCP)、PCR-限制性片段长度多态性 (RFL P)和 PCR-直接测序 (DS)技术分析 10 7株结核分枝杆菌临床分离株 emb B基因。结果 以 H3 7Rv标准株为对照 ,10 7株结核分枝杆菌临床分离株的 16 S r DNA SSCP电泳图谱均与结核分枝杆菌标准相同。 38株药物敏感株的 emb B基因、SSCP均泳动正常 ,RFL P和 DS分析与对照株相同。 6 9株耐乙胺丁醇 (EMB)分离株中 ,2 5株 (36 .2 % ) emb B基因 SSCP泳动异常 ;8株 RFL P分析异常 ;DS分析 2 5株均为 30 6位密码子突变 ,其中 1株合并有 2 74位 CGC→ CCC突变 ,其 EMB MICs均≥ 2 0 μg/ml;8株为 30 6位 ATG→ATA或 ATT突变 ,17株为 ATG→GTG或 CTG突变 ,后者 EMB MICs均≥ 30μg/ml。结论 部分结核分枝杆菌耐乙胺丁醇是由于其 emb B基因 (尤其是 30 6位密码子 )突变所致 ,PCR- SSCP技术可能成为测定部分结核分枝杆菌乙胺丁醇耐药基因型的简便。

ObjectiveTo study the molecular mechanisms of ethambutol resistance in Mycobacterium tuberculosis, and develope a new method for detecting drug resistance. Method107 clinical isolates were identified with their microbacterial species, and then analyzed their embB genes with PCR-SSCP, PCR-RFLP and PCR-Direct Sequencing. ResultMycobacterium tuberculosis strain H 37R V was used as a control. 107 clinical isolates all had the same 16S rDNA SSCP profiles as M.tuberculosis. 38 drug-sensitive isolates had normal embB SSCP and RFLP profiles. Of 69 ethambutol-resistant isolates, 25(36.2%) displayed abnormal embB SSCP profiles; 8 isolates had abnormal RFLP profiles. All of embB mutations situated at codon 306 (1 of these had another mutation at codon 274), which EMB MICs were more than 20μg/ml. 8 isolates had ATG to ATA or ATT mutations at codon 306; 17 isolates had ATG to GTG or CTG mutations at codon 306, which EMB MICs were more than 30μg/ml. ConclusionEthambutol resistance in some M.tuberculosis isolates were due to mutations on embB genes. PCR-SSCP method might become a simple and rapid diagnostic test for genotypes of M.tuberculosis ethambutol-resistance.