探针熔解分析法快速检测结核分枝杆菌乙胺丁醇耐药突变

目的 评价探针熔解分析法快速检测结核分枝杆菌乙胺丁醇耐药突变的应用价值,为临床检测提供指导依据.方法 613份痰标本于2009年9月至2010年4月收集自厦门市疾病预防控制中心、厦门市第一医院和漳州市疾病预防控制中心.结核分枝杆菌H37Rv标准株来源于国家结核病参比实验室.37株包含结核与非结核分枝杆菌的标准盘由中国药品生物制品检定所提供.首先采用梯度稀释的野生型标准株H37Rv DNA考察探针熔解曲线分析法的分析灵敏度,然后用标准盘验证其检测特异性,最后以测序法为对照方法,采用613份结核分枝杆菌的标本评价该方法的临床应用价值.结果 探针熔解分析法可检测到3拷贝/反应,且能特异检测结核分枝杆菌.613份结核分枝杆菌的检测结果显示,符合要求的583份标本的试剂盒检测结果与测序结果完全一致,其中embB306突变菌株数为34株,embB 378-380突变菌株数为23株,embB 406突变菌株数为3株,embB 497突变菌株数为3株.结论 探针熔解分析法检测结核分枝杆菌乙胺丁醇耐药突变特异性好,灵敏度高,能有效地检测临床标本常见乙胺丁醇耐药突变位点,是值得推广的快速检测方法.

Abstract：

Objective To evaluate the potential use of a probe melting analysis (PMA) assay in detecting the embB mutations which confer resistance against ethambutol in Mycobacterium tuberculosis. Methods The analysis sensitivity and specificity of PMA were investigated by detecting a serially diluted H37 Rv DNA and a reference panel from National Institute for the Control of Pharmaceutical and Biological Product. Six hundred and thirteen sputum samples were collected from the Xiamen Center for Disease Control and Prevention, Xiamen First Hospital and Center for Zhangzhou Disease Control and Prevention from September 2009 to April 2010. The PMA assay was then evaluated by detecting 613 clinical isolates and the results were compared with the sequencing results. Results The PMA assay could specifically detect Mycobacterium tuberculosis and had a limit of detection of 3 copies per reaction. The assay results with 613 clinical isolates showed that PMA gave a 100% concordance with sequencing in the 583 qualified samples, among which 34 were mutations at embB 306,23 at embB 378-380, 3 at embB 406 and 3 at embB 497. Conclusions PMA assay is a sensitive and specific method enabling efficient detection of common embB mutations causing ethambutol-resistance. The rapidness of this method together with its reliability would facilitate its use in routine testing.

Probe melting analysis for rapid detection of ethambutol-resistant mutations in Mycobacterium tuberculosis clinical isolates

Objective To evaluate the potential use of a probe melting analysis (PMA) assay in detecting the embB mutations which confer resistance against ethambutol in Mycobacterium tuberculosis. Methods The analysis sensitivity and specificity of PMA were investigated by detecting a serially diluted H37 Rv DNA and a reference panel from National Institute for the Control of Pharmaceutical and Biological Product. Six hundred and thirteen sputum samples were collected from the Xiamen Center for Disease Control and Prevention, Xiamen First Hospital and Center for Zhangzhou Disease Control and Prevention from September 2009 to April 2010. The PMA assay was then evaluated by detecting 613 clinical isolates and the results were compared with the sequencing results. Results The PMA assay could specifically detect Mycobacterium tuberculosis and had a limit of detection of 3 copies per reaction. The assay results with 613 clinical isolates showed that PMA gave a 100% concordance with sequencing in the 583 qualified samples, among which 34 were mutations at embB 306,23 at embB 378-380, 3 at embB 406 and 3 at embB 497. Conclusions PMA assay is a sensitive and specific method enabling efficient detection of common embB mutations causing ethambutol-resistance. The rapidness of this method together with its reliability would facilitate its use in routine testing.