Loop-mediated isothermal amplification for direct detection of Mycobacterium tuberculosis complex, M-avium, and M-intracellulare in sputum samples
Loop-mediated isothermal amplification for direct detection of Mycobacterium tuberculosis complex, M-avium, and M-intracellulare in sputum samples
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DOI：
10.1128/jcm.41.6.2616-2622.2003
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发表时间：
2003-06-01
影响因子：
9.4
通讯作者：
Hayashi, K
中科院分区：
文献类型：
--
作者：
Iwamoto, T;Sonobe, T;Hayashi, K
环介导的等温扩增(LAMP)是一种新型的核酸扩增方法，它是一种试剂在等温条件下进行反应的新方法，具有高特异性、高效和快速的特点。我们使用LAMP直接从痰标本中检测结核分枝杆菌复合体、鸟型分枝杆菌和细胞内分枝杆菌，以及检测在液体培养基(MGIT；日本贝顿·迪金森有限公司，日本东京)或固体培养基(小川氏培养基)上生长的培养菌株。针对gyrB基因设计了种属特异性引物，并在24种分枝杆菌和7种非分枝杆菌上验证了它们的特异性。整个过程非常简单，首先在一个试管中混合所有试剂，然后进行等温反应，在此期间反应混合物保持在63摄氏度。通过在反应管中加入SYBR Green I，可以直观地显示得到的扩增产物。扩增反应所需的唯一设备是常规的实验室水浴或加热块，提供63摄氏度的恒温。该方法的检测下限为5-50个拷贝的纯化DNA，孵育时间为60分钟。用固体培养法鉴定结核分枝杆菌、禽分枝杆菌和胞内分枝杆菌，反应时间可缩短至35min。对66份痰标本中用于Amplicor试验(罗氏诊断公司)的残留DNA裂解产物进行了LAMP试验。尽管后一种方法的样本量很小，只有2.75毫升的DNA裂解物，但它的性能与需要50毫升DNA裂解物的Amplicor法相当。这种基于LAMP的分析方法简单、快速、灵敏；固体培养在35分钟内就能得到结果，液体培养在60分钟内就能得出结果，或者对于含有可用于检测的相应数量DNA的痰标本。
Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method in which reagents react under isothermal conditions with high specificity, efficiency, and rapidity. We used LAMP for detection of Mycobacterium tuberculosis complex, Mycobacterium avium, and Mycobacterium intracellulare directly from sputum specimens as well as for detection of culture isolates grown in a liquid medium (MGIT; Nippon Becton Dickinson Co., Ltd., Tokyo, Japan) or on a solid medium (Ogawa's medium). Species-specific primers were designed by targeting the gyrB gene, and their specificities were validated on 24 mycobacterial species and 7 nonmycobacterial species. The whole procedure is quite simple, starting with the mixing of all reagents in a single tube, followed by an isothermal reaction during which the reaction mixture is held at 63degreesC. The resulting amplicons are visualized by adding SYBR Green I to the reaction tube. The only equipment needed for the amplification reaction is a regular laboratory water bath or heat block that furnishes a constant temperature of 63degreesC. The assay had a detection limit of 5 to 50 copies of purified DNA with a 60-min incubation time. The reaction time could be shortened to 35 min for the species identification of A tuberculosis complex, M. avium, and M. intracellulare from a solid-medium culture. Residual DNA lysates prepared for the Amplicor assay (Roche Diagnostics GmbH) from 66 sputum specimens were tested in the LAMP assay. Although the sample size used for the latter assay was small, 2.75 mul of the DNA lysates, it showed a performance comparable with that of the Amplicor assay, which required 50 mul of the lysates. This LAMP-based assay is simple, rapid, and sensitive; a result is available in 35 min for a solid-medium culture and in 60 min for a liquid-medium culture or for a sputum specimen that contains a corresponding amount of DNA available for testing.