著者

桝田 和彌 青木 萌 寺澤 沙希 飯野 久和 Kazuya MASUDA Megumi AOKI Saki TERASAWA Hisakazu IINO

出版者

昭和女子大学近代文化研究所

雑誌

学苑 = Gakuen (ISSN:13480103)

巻号頁・発行日

no.938, pp.26-31, 2018-12-01

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Recently, studies of intestinal microbiota have been conducted using mainly next-generation sequencers to perform comprehensive bacterial DNA analyses. When using this molecular biological approach, intestinal bacterial DNA is extracted from fecal samples. But the influence of the fecal sample storage condition and the methods of DNA extraction on the analysis have not been investigated as far as we know. In this study, we evaluate the effects of different freezing conditions and storage periods of microbial DNA in fecal samples using PCR-DGGE analysis. Fecal samples were stored at −20 ºC, −80 ºC and −80 ºC followed by a liquid nitrogen treatment and kept for 3 months and 1 year, respectively. Microbial DNA extracted from these fecal samples was examined using PCR-DGGE analysis to monitor total intestinal microbiota: Bacteroides, Bifidobacterium, Lactobacillus and Clostridium groups. DGGE profiles demonstrated that the total bacterial flora was stable and no significant changes were found due to storage conditions or periods. In genus specific detection of samples stored for three months, DNA bands were detected in all samples except for in part of the Clostridium group. In the case of fecal samples stored for one year, both at −80 ºC and also treated with liquid nitrogen, amplified genus specific bands were present in all samples. A different band pattern was observed only in the amplicon of the liquid nitrogen treated samples from the Clostridium group. On the other hand, in microbial DNA extracted from samples preserved at −20 ºC it was impossible to amplify specific fragments. Since some bacterial groups in fecal samples were affected by the freezing method, storage conditions and period, it appears that rapidly freezing fecal samples may be the most effective way to maintain intestinal microbiota.