A novel and more sensitive loop-mediated isothermal amplification assay targeting IS6110 for detection of Mycobacterium tuberculosis complex
A novel and more sensitive loop-mediated isothermal amplification assay targeting IS6110 for detection of Mycobacterium tuberculosis complex
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DOI：
10.1016/j.micres.2009.05.001
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发表时间：
2010-01-01
影响因子：
6.7
通讯作者：
Romano, Marta
中科院分区：
文献类型：
--
作者：
Aryan, Ehsan;Makvandi, Manoochehr;Romano, Marta
开发改进的结核病诊断方法是国际研究的优先事项之一，因为结核病仍然是全球主要的健康威胁。环介导的等温扩增(LAMP)是一种新的核酸检测方法，它不需要昂贵或复杂的仪器，可以在低资源环境下使用。利用重复插入序列IS6110作为靶基因，我们建立了一种高效的LAMP方法，该方法可以特异性地检测结核分枝杆菌复合体(MTBC)的成员。结果表明，这种方法比基于IS6110的常规聚合酶链式反应的敏感性高20倍。此外，它的灵敏度分别比之前描述的两种基于gyrB和RRS的结核分枝杆菌LAMP检测方法高出50倍和20倍。LAMP和套式聚合酶链式反应的敏感性相同，但LAMP检测比后者更快速、更经济。虽然，我们的LAMP检测可以使用非变性模板成功地进行，但这导致检测的灵敏度降低了200倍。此外，通过对15例结核病患者的临床痰标本进行LAMP检测，我们能够检测出结核分枝杆菌。综上所述，我们的初步结果表明，基于IS6110的MTBC-LAMP方法是一种很有前途的新的结核病诊断方法，具有很高的灵敏度，可以很容易地应用于低资源环境下的结核病诊断。(C)2009年爱思唯尔股份有限公司。版权所有。
Developing improved tuberculosis (TB) diagnostics is one of the international research priorities, as TB remains globally a major health threat. Loop-mediated isothermal amplification (LAMP) is a new nucleic acid detection method that can be used in low-resource settings, because it does not require expensive or complex instruments. Using the repetitive insertion sequence IS6110 as a target gene, we developed an efficient LAMP assay, which specifically detects members of the Mycobacterium tuberculosis complex (MTBC). This assay proved 20 times more sensitive than IS6110-based conventional PCR. Moreover, its sensitivity was, respectively, 50 and 20 times higher than the one obtained with the two previously described LAMP assays for M. tuberculosis, based on gyrB and rrs, respectively. Identical sensitivities were obtained for LAMP and nested PCR, but the LAMP assay was more rapid and cost-effective than the latter. Although, our LAMP assay can successfully be performed using a non-denatured template, this results in a 200-fold reduction in the sensitivity of the assay. Moreover, by performing our LAMP assay on 15 clinical sputum samples from TB patients we were able to detect MTB. Taken together, our preliminary results indicate that IS6110-based MTBC-LAMP assay is a promising new TB-diagnostic test, with high sensitivity and that could easily be applied for the diagnosis of TB in a low-resource setting. (C) 2009 Elsevier GmbH. All rights reserved.