Kawada Yuki, Naito Yuji, Andoh Akira, Ozeki Motoyuki, Inoue Ryo
Laboratory of Animal Science, Department of Agriculture and Life Science, Kyoto Prefectural University, 1-5 Hangi-cho, Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan.
Department of Molecular Gastroenterology and Hepatology, Kyoto Prefectural University of Medicine, Kajii-cho, Kamigyo-ku, Kyoto 602-8566, Japan.
J Clin Biochem Nutr. 2019 Mar;64(2):106-111. doi: 10.3164/jcbn.18-84. Epub 2018 Dec 13.
The effect of two factors, storage and the bacterial DNA extraction method, that potentially affect the 16S rRNA-based profiling of the microbiota in the feces of Japanese adults, were evaluated. Profiles of the microbiota in feces stored in DESS (DMSO-EDTA-salt solution) for 1, 2 and 3 weeks at room temperature, and for 3 weeks at 4°C were compared with those in fresh feces and feces stored in guanidine thiocyanate solution for 3 weeks at 4°C. None of the storage variables (preservation solution, temperature and duration) considerably affected α- and β-diversity of the fecal microbiota and OTU profiles. Regarding the bacterial DNA extraction methods, four were evaluated; A) silica membrane DNA purification combined with bead-beating bacterial disruption, B) magnetic bead DNA purification combined with bead-beating bacterial disruption, C) manual DNA purification using phenol-chloroform and ethanol precipitation combined with enzymatic bacterial lysis, and D) DNA extraction by a commercially available DNA stool kit. While methods A, B, and C did not markedly affect α- and β-diversity of the fecal microbiota and the OTU profiles, method D noticeably altered both α- and β-diversity. In addition, method D caused significant changes in the abundance of two predominant genera; and .
评估了储存和细菌DNA提取方法这两个可能影响日本成年人粪便中基于16S rRNA的微生物群分析的因素的作用。将在室温下于DESS（二甲基亚砜-乙二胺四乙酸-盐溶液）中储存1、2和3周以及在4°C下储存3周的粪便中的微生物群谱，与新鲜粪便以及在4°C下于硫氰酸胍溶液中储存3周的粪便中的微生物群谱进行比较。没有一个储存变量（保存溶液、温度和持续时间）对粪便微生物群的α和β多样性以及OTU谱有显著影响。关于细菌DNA提取方法，评估了四种方法；A）硅胶膜DNA纯化结合珠磨法破坏细菌，B）磁珠DNA纯化结合珠磨法破坏细菌，C）使用苯酚-氯仿和乙醇沉淀结合酶解细菌裂解的手动DNA纯化，以及D）通过市售的粪便DNA提取试剂盒进行DNA提取。虽然方法A、B和C对粪便微生物群的α和β多样性以及OTU谱没有明显影响，但方法D显著改变了α和β多样性。此外，方法D导致两个主要菌属的丰度发生了显著变化；以及。
