Sharma G, Tewari R, Dhatwalia S K, Yadav R, Behera D, Sethi S
Department of Microbial Biotechnology, Panjab University, Chandigarh, India.
Department of Biophysics, Panjab University, Chandigarh, India.
Lett Appl Microbiol. 2019 Mar;68(3):219-225. doi: 10.1111/lam.13115.
Quantitated Mycobacterium tuberculosis (M.tb) H37Rv DNA was used to analyse the sensitivity and the specificity was assessed using DNA isolated from the reference strain H37Rv, 12 nontuberculous mycobacterium (NTM) species and five nonmycobacterium species. Furthermore, performance of the assay was evaluated on the sputum samples and compared with smear microscopy, culture and PCR. mpt64 (also called mpb64 or Rv1980c) loop-mediated isothermal amplification (LAMP) successfully detected 1 pg DNA within 40 min and successfully rejected NTMs and other bacterial species tested. It specifically detected all the 119 confirmed TB cases and 100 of the 104 control cases. The resulting sensitivity and specificity of LAMP assay was found to be 100% (95% CI: 96·79-100%) and 96·15% (95% CI; 90·44-98·94%) respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: Loop-mediated isothermal amplification (LAMP) is a technique for isothermal DNA amplification suitable for cost-limited settings as it prevents the use of sophisticated instruments. Using mpt64 antigenic protein gene, we developed a LAMP assay especially for organisms of the M. tuberculosis complex. mpt64 LAMP assay showed 100% sensitivity and detected all the bacteriologically and clinically positive TB cases not detected by smear, culture or PCR methods.
定量的结核分枝杆菌（M.tb）H37Rv DNA用于分析敏感性，并使用从参考菌株H37Rv、12种非结核分枝杆菌（NTM）和5种非分枝杆菌中分离的DNA评估特异性。此外，在痰标本上评估该检测方法的性能，并与涂片显微镜检查、培养和PCR进行比较。mpt64（也称为mpb64或Rv1980c）环介导等温扩增（LAMP）在40分钟内成功检测到1 pg DNA，并成功排除了所检测的NTM和其他细菌种类。它特异性地检测了所有119例确诊的结核病病例和104例对照病例中的100例。发现LAMP检测的敏感性和特异性分别为100%（95%CI：96.79-100%）和96.15%（95%CI；90.44-98.94%）。研究的意义和影响：环介导等温扩增（LAMP）是一种用于等温DNA扩增的技术，适用于成本有限的环境，因为它无需使用精密仪器。利用mpt64抗原蛋白基因，我们开发了一种专门针对结核分枝杆菌复合群生物体的LAMP检测方法。mpt64 LAMP检测显示出100%的敏感性，检测出了所有涂片、培养或PCR方法未检测到的细菌学和临床阳性结核病病例。
