Department of Molecular Biology, Faculty of Natural Sciences, Comenius University, Bratislava, Slovakia.
Department of Microbiology, Molecular Biology and Biotechnology, National Agricultural and Food Centre, Food Research Institute, Bratislava, Slovakia.
J Microbiol Methods. 2021 May;184:106210. doi: 10.1016/j.mimet.2021.106210. Epub 2021 Mar 25.
Numerous factors associated with sample preparation, DNA extraction, primer choice, sequencing platform and data analysis can affect the accuracy of 16S rRNA sequencing results. The DNA extraction method is considered critical for the success of sequencing as it can be the source of considerable variations in the analysis of the microbiome. In this study, the impact of various DNA extraction methods on the results of analysis of bacterial communities in cheese was evaluated. DNA was isolated from Mozzarella as a model cheese using optimized bead-based homogenization followed by different extraction procedures. Five commercial kits and two open-formula DNA extraction protocols were evaluated for amplicon sequencing of a 16S rRNA fragment of ~1460 bp. In addition, model cheese samples artificially contaminated by defined concentrations of Listeria monocytogenes and Escherichia coli, as representatives of Gram positive and Gram negative bacteria, were analysed. Six out of seven DNA extraction procedures were found to be able to provide amplifiable bacterial DNA suitable for 16S rRNA sequence analysis, but individual extraction procedures led to variable results. In particular, lysis supported with bead-beating led to a higher proportion of G+ bacteria in relative abundance profiles, probably because of the more efficient cell wall disruption. Artificially added bacterial species were reliably detected with a quantitative response. The results demonstrated a risk in comparing the data on bacterial communities in cheese when different DNA extraction protocols are used and highlighted the need to choose a standardized approach when comparison across multiple sequencing runs is required.
许多与样品制备、DNA 提取、引物选择、测序平台和数据分析相关的因素都会影响 16S rRNA 测序结果的准确性。DNA 提取方法被认为是测序成功的关键，因为它可能是微生物组分析中产生大量差异的来源。在这项研究中，评估了各种 DNA 提取方法对奶酪中细菌群落分析结果的影响。使用优化的珠基均化法提取马苏里拉奶酪的 DNA，然后采用不同的提取方法。评估了 5 种商用试剂盒和 2 种开放式 DNA 提取方案，用于扩增约 1460 bp 的 16S rRNA 片段的扩增子测序。此外，还分析了人工添加了特定浓度李斯特菌和大肠杆菌的模型奶酪样品，它们分别是革兰氏阳性菌和革兰氏阴性菌的代表。结果发现，6 种 DNA 提取方法中有 6 种能够提供适合 16S rRNA 序列分析的可扩增细菌 DNA，但个别提取方法会导致结果存在差异。特别是，用珠磨法支持的裂解会导致相对丰度图谱中革兰氏阳性菌的比例更高，这可能是因为细胞壁的破坏更有效。人工添加的细菌物种可以通过定量反应可靠地检测到。研究结果表明，当使用不同的 DNA 提取方案时，比较奶酪中细菌群落的数据存在风险，并且当需要跨多个测序运行进行比较时，需要选择标准化的方法。
