高分辨率熔解曲线分析技术检测结核分枝杆菌的耐药性

目的:探讨高分辨率熔解曲线分析(High resolution melting,HRM)技术检测结核分枝杆菌耐药突变位点的可行性。方法:对218株结核分枝杆菌进行利福平(RFP)和异烟肼(INH)的药物敏感性测定,并进行耐药基因位点的PCR扩增和测序,同时采用HRM方法检测RFP和INH耐药基因位点情况,分析HRM的敏感性和特异性。结果:218株结核分枝杆菌药敏试验结果显示,有106株(48.6%)对RFP耐药,100株(45.9%)对INH耐药,81株(37.4%)对RFP和INH均耐药。测序发现,101株(46.3%)存在RFP耐药基因的突变,107株(49.1%)存在INH耐药基因的突变。HRM检测结果显示,100株(45.9%)存在RFP耐药基因的突变,103株(47.2%)存在INH耐药基因的突变。分别以药敏试验和测序结果为标准,HRM检测RFP耐药的敏感性为94.3%(100/106)和99.0%(100/101);特异性为97.3%(109/112)和100%(117/117);INH耐药的敏感性为97.0%(97/100)和98.1%(103/105);特异性为97.3%(109/112)和100%(113/113)。结论:HRM快速检测结核分枝杆菌耐药具有较高的特异性和灵敏度,能够满足临床需求。

Detection of Multidrug-resistant Mycobacterium Tuberculosis by High Resolution Melting Analysis of Multidrug-resistant by High Resolution Melting Analysis

Objective:To explore the feasibility in the mutation sites of Mycobacterimun tberculosis resistance by the method of high resolution melting (HRM).Methods:218 strains of Mycobacterium tuberculosis were collected to complete the drug sensibilities test of rifampin (RFP) and isoniazid (INH). The drug resistant sites were amplified by PCR and sequenced. HRM method was performed to detect the mutation sites of RFP and INH which leads to mycobacterium tuberculosis resistance, and to analyze the sensitivity and specificity of HRM method.Results:There were 106 (48.6%) RFP resistant strains, 100 (45.9%) INH resistant strains, 81 (37.4%) both RFP and INH resistant strains in drug sensibilities of 218 strains mycobacterium tuberculosis. We found 101 (46.3%) and 100 (45.9%) strains of RFP resistant gene mutation, 107 (49.1%) and 103 (47.2%) strains of INH resistant gene mutation by sequencing and HRMmethod respectively. Compared against the results of drug sensitive test and sequencing method, for detecting RFP resistance, the sensitivity of HRMwere 94.3% (100/106) and 99.0% (100/101), and specificity were 97.3% (109/112) and 100% (117/117). For detecting INH resistance, the sensitivity of HRMwere 97.0% (97/100) and 98.1% (103/105), and specificity were 97.3% (109/112) and 100%(113/113).Conclusion:The detection of resistance Mycobacterium tuberculosis by HRMmethod has a higher sensitivity and specificity to meet the clinical application.