Use of 16S rRNA Gene-Targeted Group-Specific Primers for Real-Time PCR Analysis of Predominant Bacteria in Human Feces

Abstract

16S rRNA gene-targeted group-specific primers were designed and validated for specific detection and quantification of the Clostridium leptum subgroup and the Atopobium cluster. To monitor the predominant bacteria in human feces by real-time PCR, we used these specific primers together with four sets of group-specific primers for the Clostridium coccoides group, the Bacteroides fragilis group, Bifidobacterium , and Prevotella developed in a previous study (T. Matsuki, K. Watanabe, J. Fujimoto, Y. Miyamoto, T. Takada, K. Matsumoto, H. Oyaizu, and R. Tanaka, Appl. Environ. Microbiol. 68: 5445-5451, 2002). Examination of DNA extracted from the feces of 46 healthy adults showed that the C. coccoides group was present in the greatest numbers (log 10 10.3 ± 0.3 cells per g [wet weight] [average ± standard deviation]), followed by the C. leptum subgroup (log 10 9.9 ± 0.7 cells per g [wet weight]), the B. fragilis group (log 10 9.9 ± 0.3 cells per g [wet weight]), Bifidobacterium (log 10 9.4 ± 0.7 cells per g [wet weight]), and the Atopobium cluster (log 10 9.3 ± 0.7 cells per g [wet weight]). These five bacterial groups were detected in all 46 volunteers. Prevotella was found in only 46% of the subjects at a level of log 10 9.7 ± 0.8 cells per g (wet weight). Examination of changes in the population and the composition of the intestinal flora for six healthy adults over an 8-month period revealed that the composition of the flora of each volunteer remained stable throughout the test period.