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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。
Objective: To investigate the feasibility of using high resolution melting (HRM) technique to detect drug-resistant mutations in Mycobacterium tuberculosis. Methods: 218 strains of Mycobacterium tuberculosis were assayed for the susceptibility of rifampin (RFP) and isoniazid (INH), and PCR amplification and sequencing of drug-resistant gene loci were performed. HRP was also used to detect RFP and INH resistance gene locus situation, analyze the sensitivity and specificity of HRM. Results: The susceptibility tests of 218 strains of Mycobacterium tuberculosis showed that 106 strains (48.6%) were resistant to RFP, 100 (45.9%) were resistant to INH and 81 (37.4%) were resistant to both RFP and INH . Sequencing revealed that there were mutations of RFP resistance gene in 101 strains (46.3%) and mutations of INH resistance gene in 107 strains (49.1%). The results of HRM showed that there were mutations of RFP resistance gene in 100 strains (45.9%) and mutations of INH resistance gene in 103 strains (47.2%). The sensitivity of HRM for detecting RFP resistance was 94.3% (100/106) and 99.0% (100/101) respectively, with specificity of 97.3% (109/112) and 100% ( 117/117); the sensitivity of INH resistance was 97.0% (97/100) and 98.1% (103/105); the specificity was 97.3% (109/112) and 100% (113/113). Conclusion: HRM rapid detection of Mycobacterium tuberculosis has high specificity and sensitivity, which can meet the clinical needs.