The currently used commercial DNA-extraction methods give different results of clostridial and actinobacterial populations derived from human fecal samples
The currently used commercial DNA-extraction methods give different results of clostridial and actinobacterial populations derived from human fecal samples
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DOI：
10.1111/j.1574-6941.2011.01257.x
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发表时间：
2012-03-01
影响因子：
4.2
通讯作者：
Saarela, Maria
中科院分区：
文献类型：
--
作者：
Maukonen, Johanna;Simoes, Catarina;Saarela, Maria
最近，几项与人类健康相关的微生物群研究得出了部分相互矛盾的结果。由于一些差异可能由所应用的方法解释，我们评估了不同的储存条件和常用的DNA提取试剂盒如何影响人类粪便微生物群的细菌组成、多样性和数量。根据我们的结果，DNA提取不影响拟杆菌属的多样性、组成或数量，而在-20 ℃下储存一周后，拟杆菌属的数量增加。降低1.62.5log单位（P < 0.05）。此外，在机械DNA提取后，优势细菌直肠真杆菌（Erec）组、纤细梭菌组、双歧杆菌和变形菌组的数量高0.54个对数单位（P < 0.05），如用qPCR检测的，与储存无关。此外，不同的DNA提取后，Erec组的细菌组成有显着差异;酶促DNA提取后，检测到的最普遍的属是罗斯拜瑞氏菌属（39%的克隆）和粪球菌属（10%），而机械DNA提取后，最普遍的属是布劳特氏菌属（30%），粪球菌属（13%），和Dorea（10%）。根据我们的研究结果，严格的机械裂解能够从人类粪便样品中检测出更高的细菌数量和多样性。由于梭菌和放线菌种群的结果高度依赖于所应用的DNA提取方法，因此使用不同的DNA提取方案可以解释先前获得的矛盾结果。
Recently several human health-related microbiota studies have had partly contradictory results. As some differences may be explained by methodologies applied, we evaluated how different storage conditions and commonly used DNA-extraction kits affect bacterial composition, diversity, and numbers of human fecal microbiota. According to our results, the DNA-extraction did not affect the diversity, composition, or quantity of Bacteroides spp., whereas after a week's storage at -20 degrees C, the numbers of Bacteroides spp. were 1.62.5 log units lower (P < 0.05). Furthermore, the numbers of predominant bacteria, Eubacterium rectale (Erec)-group, Clostridium leptum group, bifidobacteria, and Atopobium group were 0.54 log units higher (P < 0.05) after mechanical DNA-extraction as detected with qPCR, regardless of storage. Furthermore, the bacterial composition of Erec-group differed significantly after different DNA-extractions; after enzymatic DNA-extraction, the most prevalent genera detected were Roseburia (39% of clones) and Coprococcus (10%), whereas after mechanical DNA-extraction, the most prevalent genera were Blautia (30%), Coprococcus (13%), and Dorea (10%). According to our results, rigorous mechanical lysis enables detection of higher bacterial numbers and diversity from human fecal samples. As it was shown that the results of clostridial and actinobacterial populations are highly dependent on the DNA-extraction methods applied, the use of different DNA-extraction protocols may explain the contradictory results previously obtained.