Development of 16S rRNA-gene-targeted group-specific primers for the detection and identification of predominant bacteria in human feces

Abstract

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.

FIG. 1.

PCR products obtained for eight species with group-specific primers. Lane M, DNA size markers (sizes [in bases] are indicated on the left); lane 1, Bacteroides fragilis NCTC 9343^T; lane 2, Bacteroides vulgatus ATCC 8424 ^T; lane 3, Prevotella melaninogenica JCM 6321; lane 4, Prevotella buccae DSM 20615; lane 5, Bifidobacterium bifidum ATCC 29521^T; lane 6, Bifidobacterium longum ATCC 157071^T; lane 7, Clostridium coccoides JCM 1395^T; lane 8, Clostridium clostridioforme JCM 1291^T; lane 9, negative control (PCR performed with g-Bfra primers and Escherichia coli ATCC 11775^T).

FIG. 2.

Detection limits of the group-specific PCR methods, as determined by using DNA extracted from pure cultured B. fragilis NCTC 9343^T. Lane M, DNA size markers (sizes [in bases] are indicated on the left); lane 1, 10⁶ cells per PCR mixture; lane 2, 10⁵ cells per PCR mixture; lane 3, 10⁴ cells per PCR mixture; lane 4, 10³ cells per PCR mixture; lane 5, 10² cells per PCR mixture; lane 6, 10 cells per PCR mixture; lane 7, 1 cell per PCR mixture; lane 8, no cells (negative control).