Department of Biological Sciences, University of Idaho, Moscow, Idaho, United States of America.
PLoS One. 2012;7(3):e33865. doi: 10.1371/journal.pone.0033865. Epub 2012 Mar 23.
DNA extraction is an essential step in all cultivation-independent approaches to characterize microbial diversity, including that associated with the human body. A fundamental challenge in using these approaches has been to isolate DNA that is representative of the microbial community sampled.
METHODOLOGY/PRINCIPAL FINDINGS: In this study, we statistically evaluated six commonly used DNA extraction procedures using eleven human-associated bacterial species and a mock community that contained equal numbers of those eleven species. These methods were compared on the basis of DNA yield, DNA shearing, reproducibility, and most importantly representation of microbial diversity. The analysis of 16S rRNA gene sequences from a mock community showed that the observed species abundances were significantly different from the expected species abundances for all six DNA extraction methods used.
CONCLUSIONS/SIGNIFICANCE: Protocols that included bead beating and/or mutanolysin produced significantly better bacterial community structure representation than methods without both of them. The reproducibility of all six methods was similar, and results from different experimenters and different times were in good agreement. Based on the evaluations done it appears that DNA extraction procedures for bacterial community analysis of human associated samples should include bead beating and/or mutanolysin to effectively lyse cells.
DNA 提取是所有非培养方法研究微生物多样性的重要步骤，包括与人体相关的微生物多样性。使用这些方法的一个基本挑战是分离出具有代表性的微生物群落 DNA。
方法/主要发现：在这项研究中，我们使用 11 个人体相关细菌物种和一个含有相同数量这些物种的模拟群落，对六种常用的 DNA 提取方法进行了统计学评估。这些方法的比较基础是 DNA 产量、DNA 断裂、重现性，最重要的是微生物多样性的代表性。对模拟群落的 16S rRNA 基因序列的分析表明，所有六种 DNA 提取方法的观察到的物种丰度与预期的物种丰度显著不同。
结论/意义：包含珠磨和/或溶菌酶的方案比没有这两种方法的方案能更好地代表细菌群落结构。六种方法的重现性相似，不同实验者和不同时间的结果都非常一致。根据所做的评估，似乎用于分析与人相关样本的细菌群落的 DNA 提取程序应包括珠磨和/或溶菌酶，以有效地裂解细胞。
