Department of Basic Veterinary Sciences, Division of Microbiology and Epidemiology, University of Helsinki, P.O. Box 66, FI-00014, Helsinki, Finland.
J Microbiol Methods. 2010 May;81(2):127-34. doi: 10.1016/j.mimet.2010.02.007. Epub 2010 Feb 19.
Several different protocols are used for fecal DNA extraction, which is an integral step in all phylogenetic and metagenomic approaches to characterize the highly diverse intestinal ecosystem. We compared four widely used methods, and found their DNA yields to vary up to 35-fold. Bacterial, archaeal and human DNA was quantified by real-time PCR, and a compositional analysis of different extracts was carried out using the Human Intestinal Tract Chip, a 16S rRNA gene-based phylogenetic microarray. The overall microbiota composition was highly similar between the methods in contrast to the profound differences between the subjects (Pearson correlations >0.899 and 0.735, respectively). A detailed comparative analysis of mechanical and enzymatic methods showed that despite their overall similarity, the mechanical cell disruption by repeated bead beating showed the highest bacterial diversity and resulted in significantly improved DNA extraction efficiency of archaea and some bacteria, including Clostridium cluster IV. By applying the mechanical disruption method a high prevalence (67%) of methanogenic archaea was detected in healthy subjects (n=24), exceeding the typical values reported previously. The assessment of performance differences between different methodologies serves as a concrete step towards the comparison and reliable meta-analysis of the results obtained in different laboratories.
有几种不同的方案可用于粪便 DNA 提取，这是所有用于描述高度多样化肠道生态系统的系统发生和宏基因组学方法的一个基本步骤。我们比较了四种广泛使用的方法，发现它们的 DNA 产量差异可达 35 倍。采用实时 PCR 定量细菌、古菌和人源 DNA，并使用基于 16S rRNA 基因的系统发生微阵列的人类肠道芯片进行不同提取物的组成分析。与供体之间的显著差异相反（皮尔逊相关系数分别为>0.899 和 0.735），这些方法之间的总体微生物群落组成非常相似。尽管它们具有总体相似性，但通过反复珠磨的机械细胞破碎显示出最高的细菌多样性，并显著提高了古菌和一些细菌（包括梭菌簇 IV）的 DNA 提取效率。通过应用机械破碎方法，在 24 名健康供体中检测到产甲烷古菌的高流行率（67%），超过了先前报道的典型值。不同方法学之间性能差异的评估是朝着比较和可靠的荟萃分析不同实验室获得的结果迈出的具体一步。
