多位点酶切联合SSCP方法检测结核杆菌inhA基因突变  

Detecting InhA Genetic Mutations in Mycobacterium Tuberculosis Isolates by Combining Single-strand Conformation Polymorphism Analysis with Restriction Endonuclease

作　　者：华正豪[1] 陈澍[1] 张文宏[1] 翁心华[1] 

机构地区：[1]复旦大学附属华山医院传染科,上海200040

出　　处：《复旦学报（医学版）》2004年第1期39-41,共3页Fudan University Journal of Medical Sciences

基　　金："十五"攻关基金(2001BA705B03)

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。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 isonoazid-resistant isolates. New missense mutation included A26E, R27K, V28A, Q32A, L54V and S72T. Conclusions: PCR-SSCP digesting with restriction endonudease Hae II technique is available for detecting inhA genetic mutations in MTB isolates. It also proves that mutations of inhA gene are diversiform.

关 键 词：多位点酶切 SSCP 联合检测 结核杆菌 inhA基因 基因突变 耐药基因 异烟肼 

分 类 号：R378.911[医药卫生—病原生物学] R394[医药卫生—基础医学]