7株结核分枝杆菌耐乙胺丁醇分离株embB基因测序分析

Sequencing of embB mutation in 7 ethambutol-resistant mycobacterium tuberculosis isolates

目的 :探讨结核分枝杆菌对乙胺丁醇 (EMB)产生耐药性的分子机制 ,建立直接快速检测结核分枝杆菌EMB药物敏感性的实验方法。方法 :采用PCR扩增技术对 7株耐乙胺丁醇结核分枝杆菌进行PCR扩增 ,扩增产物经纯化后 ,直接在ABI3 77型全自动测序仪上进行DNA测序分析。结果 :7株EMB耐药株的embB基因测序有 5株 ( 71.4% )发现点突变 ,其中Met(ATG)→Lle(ATA) 1例 ,Met(ATG)→Lle(ATC) 1例 ,Met(ATG)→Lle(ATT) 2例 ,Met(ATG)→Val(GTG) 1例 ,另外 2株EMB耐药株的embB基因测序未发现突变位点。结论 :embB3 0 6位氨基酸Met被置换是结核分枝杆菌对乙胺丁醇产生耐药性的重要机制 ,测序分析可确认耐药基因的突变位点 。

Objective: To research the molecular mechanism of M.tuberculosis resistance to Ethambutol,and to set up a method for direct,rapid detection of Ethambutol resistance gene mutations in M.tuberculosis.Methods: The piece of embB gene of 7 M.tuberculosis isolates were produced by polymerase chain reation, and then purified.The purified productions were analysed by sequencing directly.Results: 5 of 7(71 4%) EMB Ethambutol resistance embB genes showed apparent differences with genes of standard strain . and the mutation site was at codon 306 of embB. 1 Met(ATG)→Lle(ATA),1 Met(ATG)→Lle(ATC),2 Met(ATG)→Lle(ATT) and 1 Met(ATG)→Val(GTG).other 2 EMB Ethambutol-resistance embB genes didn't show abnormity.Conclusion: The codon 306 of embB gene mutation is an important mechanism of M.tuberculosis resistance to Ethambutol. DNA squencing mighe be used as a simple, rapid and reliable diagnostic method for M.tuberculosis in recently.