应用等位基因特异性扩增法快速检测结核分支杆菌对利福平的耐药性

Rapid Detection of Rifampin Resistance of Mycobacterium tuberculosis by Allele Specific Amplification

目的 建立一套等位基因特异性扩增 (ASA)的检测体系 ,用于结核分支杆菌对利福平耐药性的快速检测。方法 根据结核分支杆菌野生型基因设计ASA特异性引物 ,ASAPCR扩增 39株结核杆菌利福平耐药株的rpoB基因 ,经琼脂糖凝胶电泳检测 ,进而推断利福平的耐药性 ;同时进行DNA序列测定以验证ASA法的检测结果 ,并分析上海地区结核杆菌rpoB基因的突变特点。结果 在 39株利福平耐药株中共检出 36株 ,敏感性为92 3% ;与DNA测序结果的符合率为 87.2 % ,并鉴定了 11种不同类型的 4 1种突变形式 ,其中包括 9种点突变和 2种基因缺失。结论 用等位基因特异性扩增法分析结核分支杆菌的rpoB基因突变具有较高的特异性与敏感性。该法快速、简便、可靠 ,可应用于临床利福平耐药性的检测。

Objective To establish an allele specific amplification (ASA) syste m for the rapid detection of rifampin resistance of Mycobacterium tuberculosis . Methods Design ASA specific primers according to the gene sequence of wild typ e of Mycobacterium tuberculosis, and amplify the rpoB genes of 39 rifampin- resi stant strains by ASA PCR. Determine the PCR product by agarose gel electrophore sis and speculate its rifampin resistance. Meanwhile, verify the detection result o f ASA method by DNA sequencing, and analyze the characteristics of rpo B gene mu tation in Shanghai. Results Of the 39 rifampin-resistant strains, 36 were detected by ASA. The sensitivity of ASA method was 92.3%. The consistence rate of results by ASA and DNA sequenc ing was 87.2%. A total of 41 mutations of 11 types, including 9 point mutations and 2 gene deletions, were identified in rifampin-resistant isolates in Shangha i.Conclusion ASA method showed high sensitivity and specificity in the detection of rpoB gene mutations. The method was rapid, simple and reliable, and may be used for the c linical detection of rifampin resistance of M.tuberculosis.