Microbial diversity in fecal samples depends on DNA extraction method: easyMag DNA extraction compared to QIAamp DNA stool mini kit extraction.
Microbial diversity in fecal samples depends on DNA extraction method: easyMag DNA extraction compared to QIAamp DNA stool mini kit extraction.
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DOI：
10.1186/1756-0500-7-50
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发表时间：
2014-01-21
影响因子：
1.8
通讯作者：
Krogfelt KA
中科院分区：
文献类型：
--
作者：
Mirsepasi H;Persson S;Struve C;Andersen LO;Petersen AM;Krogfelt KA
当从标本中提取细菌DNA用于分子诊断时，存在挑战，因为粪便样本还含有来自人类细胞的DNA和来自食物、细胞残留物和药物的许多不同物质，这些物质可以抑制下游PCR。本研究的目的是评估两种不同的DNA提取方法，以选择最有效的方法研究肠道细菌的多样性，变性梯度凝胶电泳（DGGE）。在本研究中，半自动DNA提取系统（easyMag®，BioMérieux，Marcy I 'Etoile，法国）和手动DNA提取系统（QIAamp DNA Stool Mini Kit，Qiagen，希尔登，德国）对从3名炎症性肠病（IBD）患者和5名健康个体收集的粪便样品进行了测试。通过QIAamp DNA Stool Mini Kit获得的DNA提取物与通过easyMag®从相同粪便样品获得的DNA提取物相比产生更高量的DNA。此外，使用easyMag®获得的DNA提取物似乎含有抑制性化合物，因为为了进行成功的PCR分析，样品应稀释至少10倍。对QIAamp DNA Stool Mini Kit提取的DNA进行DGGE，结果表明，DGGE结果与QIAamp DNA Stool Mini Kit的结果一致。QIAamp DNA Stool Mini Kit DNA提取物是DGGE运行的最佳选择，与easyMag®相比，这种提取方法产生的DNA量更高。
There are challenges, when extracting bacterial DNA from specimens for molecular diagnostics, since fecal samples also contain DNA from human cells and many different substances derived from food, cell residues and medication that can inhibit downstream PCR. The purpose of the study was to evaluate two different DNA extraction methods in order to choose the most efficient method for studying intestinal bacterial diversity using Denaturing Gradient Gel Electrophoresis (DGGE). In this study, a semi-automatic DNA extraction system (easyMag®, BioMérieux, Marcy I’Etoile, France) and a manual one (QIAamp DNA Stool Mini Kit, Qiagen, Hilden, Germany) were tested on stool samples collected from 3 patients with Inflammatory Bowel disease (IBD) and 5 healthy individuals. DNA extracts obtained by the QIAamp DNA Stool Mini Kit yield a higher amount of DNA compared to DNA extracts obtained by easyMag® from the same fecal samples. Furthermore, DNA extracts obtained using easyMag® seemed to contain inhibitory compounds, since in order to perform a successful PCR-analysis, the sample should be diluted at least 10 times. DGGE performed on PCR from DNA extracted by QIAamp DNA Stool Mini Kit DNA was very successful. QIAamp DNA Stool Mini Kit DNA extracts are optimal for DGGE runs and this extraction method yields a higher amount of DNA compared to easyMag®.