Liu Dongxin, Zhao Bing, Ou Xichao, Zheng Huiwen, Ma Aijing, He Wencong, Wang Shengfen, Zhou Yang, Xia Hui, Zheng Yang, Hou Ping, He Guangxue, Zhao Yanlin
National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Changping, Changbai Road 155, Beijing 102206, China.
National Tuberculosis Reference Laboratory, Chinese Center for Disease Control and Prevention, Changping, Changbai Road 155, Beijing 102206, China.
J Microbiol Methods. 2018 Feb;145:59-65. doi: 10.1016/j.mimet.2017.11.003. Epub 2017 Nov 3.
Mycobacterium tuberculosis (MTB) is the causative agent of pulmonary tuberculosis. Rapid and accurate diagnosis is crucial to tuberculosis control and prevention. A series of diagnostic methods has been available for MTB detection; however, new rapid, simple and affordable methods are needed. In this study, a multiple cross displacement amplification (MCDA)-based assay was developed to detect the IS6110 gene of the M. tuberculosis complex. Hydroxy naphthol blue (HNB), a colorimetric indicator, was used to detect amplification products. Amplification was carried out at a constant temperature (68°C) for only 40min, followed by direct determination of amplification products through observation of color variations. The entire detection procedure, from processing of specimens to reading of results, required only 85min. Moreover, this assay, hereafter designated MTB-MCDA-HNB, was able to detect as little as 1pg of DNA extracted from the Bacille Calmette-Guerin (BCG) strain of Mycobacterium bovis. No cross-reaction with nontuberculous mycobacteria (NTM) species was observed. Moreover, during testing of clinical samples, the sensitivity and specificity of MCDA results were 94.7% and 92.9%, respectively, when compared to results obtained using the Xpert MTB/RIF method. Therefore, the MTB-MCDA-HNB method developed in this study holds promise for application as an effective point-of-care test to detect M. tuberculosis.
结核分枝杆菌（MTB）是肺结核的病原体。快速准确的诊断对于结核病的控制和预防至关重要。已经有一系列用于检测MTB的诊断方法；然而，仍需要新的快速、简单且经济实惠的方法。在本研究中，开发了一种基于多重交叉置换扩增（MCDA）的检测方法来检测结核分枝杆菌复合群的IS6110基因。采用比色指示剂羟基萘酚蓝（HNB）来检测扩增产物。扩增在恒温（68°C）下仅进行40分钟，然后通过观察颜色变化直接测定扩增产物。从标本处理到结果读取的整个检测过程仅需85分钟。此外，该检测方法（以下称为MTB-MCDA-HNB）能够检测到低至1 pg从牛分枝杆菌卡介苗（BCG）菌株提取的DNA。未观察到与非结核分枝杆菌（NTM）物种的交叉反应。此外，在临床样本检测中，与使用Xpert MTB/RIF方法获得的结果相比，MCDA结果的敏感性和特异性分别为94.7%和92.9%。因此，本研究中开发的MTB-MCDA-HNB方法有望作为一种有效的即时检测方法用于检测结核分枝杆菌。
