Probe melting curve analysis for rapid detection of injectable second-line drug-resistant mutations in Mycobacterium tuberculosis strains
ZHANG Ting1, FU Jun1, WANG Guo-zhi2, CHEN Bao-wen2, YANG Lei2, LU Jin-biao2, XU Ye1, LI Qing-ge1
1.The State Key Laboratory of Cellular Stress Biology, Engineering Research Center of Molecular Diagnostics,School of Life Sciences, Xiamen University, Xiamen 361102, China; 2.Division of Tuberculosis Vaccines, National Institutes for Food and Drug Control, Beijing 100050, China
Abstract:The MeltPro TB/SL assay was first evaluated by a reference panel containing 10 plasmids of different mutations to evaluate the analytical and clinical performances. The limit of detection (LOD) was studied by purified genomic DNA of a reference strain H37Rv that was serially diluted in ten-fold. The wild-type and mutant plasmids of different mutation types were mixed with serial percentages of mutation to evaluate the detection ability of mixed bacterial samples of this kit. The analytical specificity was evaluated using 23 non-tuberculous mycobacterium (NTM) isolates. The clinical performance was evaluated by analyzing 241 clinical strains. Ten plasmids of different mutations could be clearly identified from the wild-type plasmid. The LOD was 5 copies of wild-type Mycobacterium tuberculosis (MTB) genomic DNA per reaction. The assay could detect 30% mutant DNA in wild-type background. All the 23 NTM strains showed no cross reactivity with M. tuberculosis strain. Clinical study showed that the clinical sensitivity of the assay was 90.0% for kanamycin, 80.0% for amikacin, 85.7% for capreomycin, and the clinical specificity was 98.8% for kanamycin, 98.8% for amikacin, 96.6% for capreomycin, respectively. The MeltPro TB/SL assay gave a 100% concordance with sequencing analysis. The MeltPro TB/SL assay was able to reliably detect common mutations associated with injectable second-line drug-resistance. The assay has the potential to be used for rapid screening of injectable second-line drug-resistant mutations in MTB strains.
张婷, 付军, 王国治, 陈保文, 杨蕾, 卢锦标, 许晔, 李庆阁. 探针熔解曲线法快速检测结核分枝杆菌注射类二线药耐药突变[J]. 中国人兽共患病学报, 2015, 31(5): 412-417.
ZHANG Ting, FU Jun, WANG Guo-zhi, CHEN Bao-wen, YANG Lei, LU Jin-biao, XU Ye, LI Qing-ge. Probe melting curve analysis for rapid detection of injectable second-line drug-resistant mutations in Mycobacterium tuberculosis strains. Chinese Journal of Zoonoses, 2015, 31(5): 412-417.
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