The Impact of DNA Extraction Methods on Stool Bacterial and Fungal Microbiota Community Recovery
The Impact of DNA Extraction Methods on Stool Bacterial and Fungal Microbiota Community Recovery
复制标题
DOI：
10.3389/fmicb.2019.00821
复制
发表时间：
2019-04-17
影响因子：
5.2
通讯作者：
Freiberger, Tomas
中科院分区：
文献类型：
--
作者：
Fiedorova, Kristyna;Radvansky, Matej;Freiberger, Tomas
我们对健康和疾病状态下人体肠道微生物群的了解取决于准确且可重复的微生物数据获取。这个过程中的关键步骤是应用适当的方法提取微生物DNA，因为DNA提取过程中产生的偏差可能导致微生物呈现不准确。在这项研究中，我们试图找到一种可有效用于分析细菌和真菌群落的DNA提取方案。我们使用以下两种样本评估了五种DNA提取方法（QIAamp DNA Stool Mini Kit、PureLink™ Microbiome DNA Purification Kit、ZR Fecal DNA MiniPrep™ Kit、NucleoSpin® DNA Stool Kit和IHMS协议Q）对肠道细菌和真菌微生物群回收的效果：（i）添加了细菌或真菌菌株的无菌小鼠粪便的特定体系，以及（ii）未添加菌株的人类粪便。在我们的实验设置中，我们证实所检测的方法在效率和质量上存在显著差异，这影响了所鉴定的粪便微生物群组成。此外，我们的结果表明真菌DNA提取可能容易受到试剂/试剂盒污染的影响，因此在真菌组研究中应包含适当的空白对照。总体而言，国际人类微生物组联盟推荐的标准化IHMS协议Q在考虑所有分析参数时表现最佳，因此不仅可应用于细菌微生物组研究，也可应用于真菌微生物组研究。
Our understanding of human gut microbiota in health and disease depends on accurate and reproducible microbial data acquisition. The critical step in this process is to apply an appropriate methodology to extract microbial DNA, since biases introduced during the DNA extraction process may result in inaccurate microbial representation. In this study, we attempted to find a DNA extraction protocol which could be effectively used to analyze both the bacterial and fungal community. We evaluated the effect of five DNA extraction methods (QlAamp DNA Stool Mini Kit, PureLink (TM) Microbiome DNA Purification Kit, ZR Fecal DNA MiniPrep((TM)) Kit, NucleoSpir (R) DNA Stool Kit, and IHMS protocol Q) on bacterial and fungal gut microbiome recovery using (i) a defined system of germ-free mice feces spiked with bacterial or fungal strains, and (ii) non-spiked human feces. In our experimental setup, we confirmed that the examined methods significantly differed in efficiency and quality, which affected the identified stool microbiome composition. In addition, our results indicated that fungal DNA extraction might be prone to be affected by reagent/kit contamination, and thus an appropriate blank control should be included in mycobiome research. Overall, standardized IHMS protocol Q, recommended by the International Human Microbiome Consortium, performed the best when considering all the parameters analyzed, and thus could be applied not only in bacterial, but also in fungal microbiome research.