LAMP技术检测结核分枝杆菌试验性研究

Assessment of laboratory performance of Loop-Mediated Isothermal Amplification (LAMP) for detection of Mycobacterium tuberculosis

目的建立并评价环介导等温扩增技术检测结核分枝杆菌方法。方法选择临床痰标本培养的结核分枝杆菌分离物14份,非结核分枝杆菌阳性分离物14种31份,痰标本11份,以H37Rv、BCG为阳性对照,提取DNA后,用自主研发的环介导等温扩增检测技术进行检测,统计该技术对结核分枝杆菌检测的灵敏性和特异性。结果该技术对结核分枝杆菌分离物检测结果灵敏性为100%(14/14),对非结核分枝杆菌分离物检测结果特异性为100%(31/31),对人结核分枝杆菌标准菌株H37Rv、BCG检测结果均为阳性;方法的检出限为12cfu。对临床痰标本中的结核分枝杆菌检测结果与罗氏培养基培养结果一致。结论环介导等温扩增检测结核分枝杆菌技术灵敏性和特异性高,具有简便易行、快速准确的特点,适合于基层结核病诊断实验室推广应用。

Objective To set up a protocol and evaluate feasibility of LAMP for detection of Mycobacterium tuberculosis.Methods DNA were extracted from 14 Mycobacterium tuberculosis（MTB） samples and 31 non-Mycobacterium tuberculosis（NTM） samples consisted of 14 species from culture isolates grown in L-J medium,11 sputum specimens,as well as two H37Rv species and one BCG.Species-specific primers were designed by targeting the gyrB gene and a prototype LAMP assay was evaluated for accuracy and feasibility on DNA extractions.Results The sensitivity of LAMP in MTB samples was 100%（14/14）,The specificity in NTM samples was 100%（31/31）.Both H37Rv and BCG samples were LAMP positive.Results of LAMP assay for 11 sputum specimens were accordant with L-J culture.The assay had a detection limit of 1×10-4 mg/mL for either H37Rv culture isolates or BCG.Conclusions LAMP technique might provide a simple,rapid,sensitive and cost-effective platform for the molecular detection of pulmonary TB,especially in the testing centers at the grass-roots level