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Applied and Environmental Microbiology, November 2002, p. 5445-5451, Vol. 68, No. 11
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.11.5445-5451.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Development of 16S rRNA-Gene-Targeted Group-Specific Primers for the Detection and Identification of Predominant Bacteria in Human Feces

Takahiro Matsuki,1* Koichi Watanabe,1 Junji Fujimoto,1 Yukiko Miyamoto,1 Toshihiko Takada,1 Kazumasa Matsumoto,1 Hiroshi Oyaizu,2 and Ryuichiro Tanaka1

Yakult Central Institute for Microbiological Research, 1796 Yaho, Kunitachi, Tokyo 186-8650,1 Graduate School of Agriculture and Agricultural Life Science, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan2

Received 11 March 2002/ Accepted 5 August 2002

For the detection and identification of predominant bacteria in human feces, 16S rRNA-gene-targeted group-specific primers for the Bacteroides fragilis group, Bifidobacterium, the Clostridium coccoides group, and Prevotella were designed and evaluated. The specificity of these primers was confirmed by using DNA extracted from 90 species that are commonly found in the human intestinal microflora. The group-specific primers were then used for identification of 300 isolates from feces of six healthy volunteers. The isolates were clearly identified as 117 isolates of the B. fragilis group, 22 isolates of Bifidobacterium, 65 isolates of the C. coccoides group, and 17 isolates of Prevotella, indicating that 74% of the isolates were identified with the four pairs of primers. The remaining 79 isolates were identified by 16S ribosomal DNA sequence analysis and consisted of 40 isolates of Collinsella, 24 isolates of the Clostridium leptum subgroup, and 15 isolates of disparate clusters. In addition, qualitative detection of these bacterial groups was accomplished without cultivation by using DNA extracted from the fecal samples. The goal for this specific PCR technique is to develop a procedure for quantitative detection of these bacterial groups, and a real-time quantitative PCR for detection of Bifidobacterium is now being investigated (T. Requena, J. Burton, T. Matsuki, K. Munro, M. A. Simon, R. Tanaka, K. Watanabe, and G. W. Tannock, Appl. Environ. Microbiol. 68:2420-2427, 2002). Therefore, the approaches used to detect and identify predominant bacteria with the group-specific primers described here should contribute to future studies of the composition and dynamics of the intestinal microflora.

* Corresponding author. Mailing address: Yakult Central Institute for Microbiological Research, 1796 Yaho, Kunitachi, Tokyo 186-8650, Japan. Phone: 81 (42) 577 8962. Fax: 81 (42) 577 3020. E-mail: takahiro-matsuki{at}yakult.co.jp.

Applied and Environmental Microbiology, November 2002, p. 5445-5451, Vol. 68, No. 11
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.11.5445-5451.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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