应用多重PCR-单链构象多态性分析同时检测耐异烟肼和利福平的结核分枝杆菌  

A new multiplex-polymerase chain reaction-single strand conformation polymorphism assay for simultaneous detection of isoniazid and rifampin resistance in Mycobacterium tuberculosis

作　　者：程晓东[1] 杨柳[1] 刘家云[1] 岳乔红[1] 徐修礼[1] 马越云[1] 彭道荣[1] 于文彬[1] 苏明权[1] 郝晓柯[1] 

机构地区：[1]第四军医大学西京医院全军临床检验医学中心,西安710032

出　　处：《中华检验医学杂志》2008年第11期1240-1244,共5页Chinese Journal of Laboratory Medicine

摘　　要：目的探讨应用多重PCR-单链构象多态性分析（multiplex—polymerase chain reaction—single strand conformation polymorphism，multi—PCR—SSCP）方法快速、特异地同时快速检测结核分枝杆菌对异烟肼和利福平耐药性的效能。方法根据结核分枝杆菌的inhA序列、katG序列、rpoB序列，分别设计出3对特异性寡聚核苷酸引物，采用multi—PCR—SSCP技术，一次性检出耐异烟肼和利福平的结核分枝杆菌。新方法的有效性通过116株临床分离株（70株耐异烟肼，66株耐利福平）的验证。结果Multi—PCR—SSCP方法检测临床分离株基因突变的有效性，以细菌培养和药敏试验结果为金标准。116株临床分离株和H37Rv标准株中除了4株katG缺失突变，其余菌株3个基因katG、inhA和rpoB在单基因PCR中都扩增成功。与H37Rv标准株相比，46株katG基因突变，14株inhA基因突变，58株rpoB基因突变。38株katG和rpoB，4株inhA和rpoB，4株inhA和katG同时突变，还有2株3个基因都有突变。multi—PCR—SSCP对于耐异烟肼和利福平的结核分枝杆菌检出的敏感度分别为80％、82％，特异度分别为100％和92％。结论multi—PCR—SSCP方法敏感、特异，能同时快速有效地检测耐多药结核分枝杆菌，有望成为临床指导用药的好方法，为深入研究耐药基因检测奠定了良好的基础。Objective To detect the isoniazid （INH） and rifampin （RIF） resistance of Mycobacterium tuberculosis isolates in the single tube with muhiplex-polymerase chain reaction-single strand conformation polymorphism（mufti-PCR-SSCP）system. Methods According to the sequences of inhA,katG and rpoB genes of the Mycobacterium tuberculosis, three pairs of oligonucleotide primes were designed to examine the INH and RIF resistance with the muhi-PCR-SSCP. The validity of the newly developed method was evaluated with 116 clinical isolates of Mycobacterium tuberculosis （70 isolates that were INH-resistant and 66 isolates that were RIF-resistant ）. Results The validity of the method was assessed with multiplex- PCR-SSCP with the bacteria culture with susceptibility test as golden standard. The three genes, katG, inhA and rpoB, in the 116 clinical isolates and H37 Rv strain were amplified successfully in single PCR reactions, except 4 isolates with katG deletion mutants. Compared with strain H37Rv, forty-six isolates had katG gene mutations, thirteen had inhA mutations and fifty-eight had rpoB mutations. Thirty-eight isolates had simultaneous katG and rpoB mutations and 4 isolates had both inhA and rpoB mutations. Four isolates had inhA and katG mutations and 2 isolates had mutations in all three genes simultaneously. The sensitivity of the newly developed muhiplex-PCR-SSCP assay was 80% and 82% for INH and RIF, respectively. The specificity of the assay was 100% and 92% for INH and RIF, respectively. Conclusion Muhiplex-PCR- SSCP provides a rapid, specific and cost-effective method of detecting muhidrug-resistant TB. It laid a solid foundation for the further study of drug resistant gene.

关 键 词：分枝杆菌 结核 抗药性 多种 细菌 聚合酶链反应 多态现象 单链构象 

分 类 号：R686[医药卫生—骨科学]