A comparison of DNA extraction procedures for the detection of Mycobacterium ulcerans, the causative agent of Buruli ulcer, in clinical and environmental specimens
A comparison of DNA extraction procedures for the detection of Mycobacterium ulcerans, the causative agent of Buruli ulcer, in clinical and environmental specimens
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DOI：
10.1016/j.mimet.2008.10.002
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发表时间：
2009-02-01
影响因子：
2.2
通讯作者：
Portaels, Francoise
中科院分区：
文献类型：
--
作者：
Durnez, Lies;Stragier, Pieter;Portaels, Francoise
溃疡分枝杆菌是布鲁里溃疡的病原体，布鲁里溃疡是继结核病和麻风病之后的人类第三种最常见的分枝杆菌疾病。尽管该疾病与水生生态系统有关，但从环境中培养芽孢杆菌却很难实现。因此，目前的研究基于PCR检测溃疡分枝杆菌和一些密切相关的分枝杆菌中存在的插入序列IS2404。在本研究中，我们比较了四种检测溃疡分枝杆菌 DNA 的 DNA 提取方法，即一管细胞裂解和 DNA 提取程序 (OT)、FastPrep 程序 (FP)、改良的 Boom 程序 (MB) 和 Maxwell (R) 16 程序 (M16)。这些方法对溃疡分枝杆菌进行系列稀释，然后对溃疡分枝杆菌中的不同 PCR 目标进行 PCR 分析。 溃疡菌以确定每种方法的检测限 (DL)。还比较了提取的 DNA 的纯度以及所需的时间和精力。所有方法均在环境标本上进行，并在临床标本上测试了两种最佳方法（MB 和 M16），以检测溃疡分枝杆菌 DNA。在比较 DNA 提取方法的 DL 时，MB 和 M16 的 DL 显着低于 OT 和 FP。对于不同的 PCR 靶标，IS2404 显示出显着低于 mlsA、MIRU1、MIRU5 和 VNTR6 的 DL。 FP和M16比MB和OT快得多，而用MB提取的DNA的纯度显着高于用其他方法提取的DNA。 MB 在环境和临床标本上表现最佳。这项比较研究表明，改进的 Boom 程序虽然冗长，但与用于检测和鉴定临床和环境标本中溃疡分枝杆菌的其他测试方法相比，提供了更好的 DNA 提取方法。 (c) 2008 Elsevier B.V. 保留所有权利。
Mycobacterium ulcerans is the causative agent of Buruli ulcer, the third most common mycobacterial disease in humans after tuberculosis and leprosy. Although the disease is associated with aquatic ecosystems, cultivation of the bacillus from the environment is difficult to achieve. Therefore, at the moment, research is based on the detection by PCR of the insertion sequence IS2404 present in M. ulcerans and some closely related mycobacteria. In the present study, we compared four DNA extraction methods for detection of M. ulcerans DNA, namely the one tube cell lysis and DNA extraction procedure (OT), the FastPrep procedure (FP), the modified Boom procedure (MB), and the Maxwell (R) 16 Procedure (M16).The methods were performed on serial dilutions of M. ulcerans, followed by PCR analysis with different PCR targets in M. ulcerans to determine the detection limit (DL) of each method. The purity of the extracted DNA and the time and effort needed were compared as well. All methods were performed on environmental specimens and the two best methods (MB and M16) were tested on clinical specimens for detection of M. ulcerans DNA.When comparing the DLs of the DNA extraction methods, the MB and M16 had a significantly lower DL than the OT and FP. For the different PCR targets, IS2404 showed a significantly lower DL than mlsA, MIRU1, MIRU5 and VNTR6. The FP and M16 were considerably faster than the MB and OT, while the purity of the DNA extracted with the MB was significantly higher than the DNA extracted with the other methods. The MB performed best on the environmental and clinical specimens.This comparative study shows that the modified Boom procedure, although lengthy, provides a better method of DNA extraction than the other methods tested for detection and identification of M. ulcerans in both clinical and environmental specimens. (c) 2008 Elsevier B.V. All rights reserved.