Effect of DNA extraction methods and sampling techniques on the apparent structure of cow and sheep rumen microbial communities.
Effect of DNA extraction methods and sampling techniques on the apparent structure of cow and sheep rumen microbial communities.
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DOI：
10.1371/journal.pone.0074787
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Janssen PH
中科院分区：
文献类型：
--
作者：
Henderson G;Cox F;Kittelmann S;Miri VH;Zethof M;Noel SJ;Waghorn GC;Janssen PH
分子微生物生态学技术被广泛用于研究瘤胃微生物群的组成，并增加对它们所起作用的理解。因此，采样和DNA提取方法，导致足够的微生物DNA的产量，也准确地代表微生物群落是至关重要的。采用15种不同的方法从牛和羊瘤胃样品中提取DNA。DNA的产量和质量，以及其对下游PCR扩增的适用性变化很大，这取决于所使用的DNA提取方法。通过微生物标记位点的定量PCR计数进一步评价了通过这些第一质量标准的9种提取方法的DNA提取物。根据所用的DNA提取方法，在相同的瘤胃样品上测定的绝对微生物数量相差超过100倍。使用454钛焦磷酸测序法评估了相同瘤胃样品中古菌、细菌、纤毛虫原生动物和真菌群落的表观组成。微生物群落组成的显着差异，观察提取方法之间，例如在相对丰度的拟杆菌门和厚壁菌门的成员。采用一种DNA提取方法，比较了通过口服胃管或通过瘤胃瘘管从奶牛中收集的平行样品中的微生物群落，以及液体和固体瘤胃发酵组分中的微生物群落。社区代表一般相似，无论瘤胃采样技术，但显着差异，如梭菌目和甲烷短杆菌反刍动物分支的一些微生物类群的丰度进行了观察。瘤胃样品组分之间的表观微生物群落组成不同，普雷沃氏菌科在液体组分中最丰富。涉及酚-氯仿提取和机械裂解步骤的DNA提取方法往往更具可比性。然而，应避免比较来自使用不同取样技术、不同瘤胃样品组分或不同DNA提取方法的研究的数据。
Molecular microbial ecology techniques are widely used to study the composition of the rumen microbiota and to increase understanding of the roles they play. Therefore, sampling and DNA extraction methods that result in adequate yields of microbial DNA that also accurately represents the microbial community are crucial. Fifteen different methods were used to extract DNA from cow and sheep rumen samples. The DNA yield and quality, and its suitability for downstream PCR amplifications varied considerably, depending on the DNA extraction method used. DNA extracts from nine extraction methods that passed these first quality criteria were evaluated further by quantitative PCR enumeration of microbial marker loci. Absolute microbial numbers, determined on the same rumen samples, differed by more than 100-fold, depending on the DNA extraction method used. The apparent compositions of the archaeal, bacterial, ciliate protozoal, and fungal communities in identical rumen samples were assessed using 454 Titanium pyrosequencing. Significant differences in microbial community composition were observed between extraction methods, for example in the relative abundances of members of the phyla Bacteroidetes and Firmicutes. Microbial communities in parallel samples collected from cows by oral stomach-tubing or through a rumen fistula, and in liquid and solid rumen digesta fractions, were compared using one of the DNA extraction methods. Community representations were generally similar, regardless of the rumen sampling technique used, but significant differences in the abundances of some microbial taxa such as the Clostridiales and the Methanobrevibacter ruminantium clade were observed. The apparent microbial community composition differed between rumen sample fractions, and Prevotellaceae were most abundant in the liquid fraction. DNA extraction methods that involved phenol-chloroform extraction and mechanical lysis steps tended to be more comparable. However, comparison of data from studies in which different sampling techniques, different rumen sample fractions or different DNA extraction methods were used should be avoided.
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