Luna Gian Marco, Dell'Anno A, Danovaro R
Department of Marine Sciences, Marine Biology Section, Faculty of Science, Polytechnic University of Marche, Ancona, Italy.
Environ Microbiol. 2006 Feb;8(2):308-20. doi: 10.1111/j.1462-2920.2005.00896.x.
In order to evaluate whether different DNA extraction procedures can affect estimates of benthic bacterial diversity, based on 16S rRNA gene terminal restriction fragment length polymorphism (T-RFLP) fingerprinting technique, we compared two in situ lysis procedures (a SDS-based protocol and a commercial kit for DNA recovery) and one cell-extraction protocol on a variety of marine sediments. Despite the two in situ lysis procedures resulted in significantly different DNA yields (highest with the SDS in situ lysis), estimates of bacterial diversity provided a not significantly different ribotype richness, as well as similar values of the Shannon-Wiener (H') and Margalef (d) indices of biodiversity and of evenness (Pielou index, J). Conversely, the cell-extraction procedure for DNA extraction resulted always in a significantly lower ribotype richness and diversity. The analysis of similarities (anosim) among the T-RFLP electropherograms allowed concluding that ribotypes composition did not change significantly using different protocols. However, the analysis of beta-diversity (turnover diversity) revealed that a large number of ribotypes was observed exclusively with one of the three protocols utilized. When unshared ribotypes from in situ lysis and cell extraction were pooled together, total ribotype richness resulted much higher (up to 80%). Our results indicate that estimates of ribotype diversity based on a single protocol of DNA extraction can significantly underestimate the total number of bacterial ribotypes present in the benthic domain. We recommend that future studies will not only integrate different DNA extraction procedures, but also will explore the possibility of integrating two or more different genetic markers in order to increase our ability to detect the actual bacterial diversity in environmental samples.
为了评估不同的DNA提取方法是否会影响对底栖细菌多样性的估计，我们基于16S rRNA基因末端限制性片段长度多态性（T-RFLP）指纹技术，在多种海洋沉积物上比较了两种原位裂解方法（一种基于SDS的方案和一种用于DNA回收的商业试剂盒）以及一种细胞提取方案。尽管两种原位裂解方法产生的DNA产量显著不同（SDS原位裂解的产量最高），但细菌多样性估计提供的核糖体类型丰富度没有显著差异，生物多样性的香农-维纳（H'）指数、马加利夫（d）指数以及均匀度（皮洛指数，J）的值也相似。相反，用于DNA提取的细胞提取方法始终导致核糖体类型丰富度和多样性显著降低。对T-RFLP电泳图谱的相似性分析（anosim）得出结论，使用不同方案时核糖体类型组成没有显著变化。然而，β多样性（周转多样性）分析表明，仅在使用的三种方案之一中观察到大量核糖体类型。当将原位裂解和细胞提取中未共享的核糖体类型合并在一起时，总核糖体类型丰富度要高得多（高达80%）。我们的结果表明，基于单一DNA提取方案的核糖体类型多样性估计可能会显著低估底栖区域中存在的细菌核糖体类型总数。我们建议未来的研究不仅要整合不同的DNA提取方法，还要探索整合两种或更多不同遗传标记的可能性，以提高我们检测环境样品中实际细菌多样性的能力。
