A novel isothermal amplification-based method to detect Mycobacterium tuberculosis complex
A novel isothermal amplification-based method to detect Mycobacterium tuberculosis complex
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DOI：
10.1016/j.mimet.2017.11.003
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发表时间：
2018-02-01
影响因子：
2.2
通讯作者：
Zhao, Yanlin
中科院分区：
文献类型：
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作者：
Liu, Dongxin;Zhao, Bing;Zhao, Yanlin
结核分枝杆菌(MTB)是肺结核的病原体。快速、准确的诊断是结核病控制和预防的关键。已有一系列诊断方法可用于结核分枝杆菌的检测，但需要新的快速、简单和负担得起的方法。在这项研究中，建立了一种基于多重交叉置换扩增(MCDA)的检测结核分枝杆菌复合体IS6110基因的方法。用比色指示剂羟基萘酚蓝(HNB)检测扩增产物。在恒温(68℃)下仅扩增40分钟，然后通过观察颜色变化直接测定扩增产物。整个检测过程，从样品的处理到结果的读取，只需要85分钟。此外，该方法能够检测到从卡介苗(BCG)中提取的牛分枝杆菌DNA，最低可达1pg，以下简称MTB-MCDA-HNB。未观察到与非结核分枝杆菌(NTM)的交叉反应。此外，在临床样本检测中，与Xpert MTB/RIF方法相比，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方法有望成为检测结核分枝杆菌的一种有效的护理点检测方法。
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 degrees C) for only 40 min, 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 85 min. Moreover, this assay, hereafter designated MTB-MCDA-HNB, was able to detect as little as 1 pg 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.