【摘要】 目的 建立一种简便而有效的方法检测结核分枝杆菌异烟肼耐药基因inhA突变。方法 收集结核杆菌临床菌株48株,其中异烟肼敏感株25株,耐药株23株。抽提临床菌株DNA和H37Rv标准株DNA,聚合酶链反应(PCR)方法扩增inhA基因,产物纯化后用限制性内切酶Hae Ⅱ酶切,酶切片段用单链多态构象分析(SS-CP)方法检测其单链构象。发现单链构象改变的PCR产物进行DNA测序。结果 48株菌株中,25株敏感株中未见inhA基因单链构象差异,23株耐药株中inhA基因突变率为21.8％。新发现的错义突变有:A26E、R27K、V28A、Q32A、L54V和S72T。结论 应用限制性内切酶Hae Ⅱ改良的聚合酶链反应—单链多态构象分析(PCR—SSCP)技术适用于结核临床菌株inhA基因突变的筛选。inhA基因突变位点呈多样性。更多还原

【Abstract】 Purpose To detect inhA genetic mutations in Mycobacterium tuberculosis (MTB) isolates, therefore to get full mutation information. Methods A total of 48 isolates of M. tuberculosis, including 23 drug-resistant MTB isolates were collected. Their genome DNA were extracted, the targets gene were gained by PCR, the products were purified and digested with restriction endonuclease Hae Ⅱ. Single-strand conformation polymorphism (SSCP) method was used to detect mutations in these isolates. DNA analysis was used to identify mutant sequences when conformation change was found. Results No inhA mutation has been identified in isoniazid- susceptible isolates while conformation changes were found in five isoniazid-resistant isolates. The mutation rate was 21. 8% in isoni-azid-resistant isolates. New missense mutation included A26E, R27K, V28A, Q32A, L54V and S72T. Conclusions PCR-SSCP digesting with restriction endonuclease Hae Ⅱ technique is available for detecting inhA genetic mutations in MTB isolates. It also proves that mutations of inhA gene are diversiform.更多还原