结核分支杆菌rpoB基因突变与耐利福平的研究

Study of rpoB Mutation in Rifampin Resistants Mycobacterium Tuberculosis

目的 研究结核分支杆菌耐利福平 (RFP)分离株rpoB基因突变情况并评价其应用价值。方法 采用聚合酶链反应一单链构象多态性 (PCR -SSCP)方法对 3 5 5株结核分支杆菌临床分离株 (其中敏感株 10 9株 ,耐RFP或含耐RFP株 2 46株 )rpoB基因进行检测 ,并对其中 3 1株耐药株用双脱氧末端终止法进行测序分析。结果 以结核分支杆菌标准株H3 7Rv为对照 ,所有 10 9株药物敏感株rpoB基因均无突变 ;2 46株耐药株中 ,2 2 5株rpoB基因SSCP图谱异常 ,其敏感性为 91 5 %。 3 1株耐药株 (其中SSCP异常 2 5株 ,正常 6株 )PCR扩增产物经测序证实 ,19株在 5 3 1位密码子TCG -TTG ,7株 5 2 6位密码子CAC -TAC ,3株在 5 16位密码子GAC -GTC ,2株未改变。结论 rpoB基因突变是耐RFP结核分支杆菌耐药性的主要分子机制 ;其最常见的突变位点是 5 3 1位色氨酸和 5 2 6位组氨酸 ,PCR

Objective To understand the rpoB gene mutation in M.tuberculosis isolates,and to evaluate their clinical value.Method 335 clinical isolates of mycobacterium tuberculosis(109 isolates drug susceptible,246 isolates rifampin-resistance or multidrug resistance including rifampin) were detected using polymerase chain reaction-single strand conformation polymorphism(PCR-SSCP).Results SSCP pattern of reference mycobacterium tuberculosis H37Rv as control,no mutation was found in rifampin-suscepitible 109 strains.SSCP patterns of 225/246 rifampin resistant clinical isolates were different from the normal control.The sensitivity was 91 5%.31 resistant isolates,included 25 abnormal isolates and 6 normal isolates of SCCP were identified.Sequencing showed 29 isolates had rpoB gene mutations and 3 isolates were not found rpoB gene mutations.The 3 most frequent rpoB gene mutation situs were Leu-531(19 isolates.TCGTTG) and His-526(7 isolates,CACTAC) and Asp-516(3 isolates,GACGTC).Conclusions The results confirm that rpoB gene mutation is the most important mechanism in rifampin resistant tuberculosis mutation situs are 531 tryptophan and 526 histidine;respectively.It is feasible that using PCR-SSCP to detect drug resistance in mycobacterium tuberculosis.