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 groupspecific 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

Long-term ecological impacts of antibiotic administration on the human intestinal microbiota

“…The 16S rRNA genes in the extracted DNA were amplified using the general eubacterial primers fD1-FAM (Weisburg et al, 1991) labelled at the 5 0 end with FAM fluorescein, and 926r (Muyzer et al, 1993). For amplification of Bacteroides spp., the reverse primer g-Bfra-R (Matsuki et al, 2002) was used together with fD1-FAM (for primer sequences, see Table 1). The PCR products were digested with HaeIII and the fluorescent terminal restriction fragments (TRFs) were separated according to size and quantified on an ABI sequencer as described previously (Jernberg et al, 2005).…”

Molecular analysis of the gut microbiota of identical twins with Crohn's disease

“…In addition, 16S rRNA genes of the B. fragilis subgroup were specifically PCR amplified using a B. fragilis subgroup-specific reverse primer, g-Bfra-R (5 0 -CCAGTATCAACTGCAATTTTA-3 0 ) (Matsuki et al, 2002) in combination with the same endlabeled Bact-8F general bacterial forward primer mentioned above. PCR amplification was carried out with an initial denaturation step at 95 1C for 3 min, followed by 30 cycles consisting of 20 s at 95 1C, 20 s at 49 1C and 30 s at 72 1C.…”

Influence of the Milk Formula Enriched With Bifidobacteria Strain Bb12 on Development of Intestinal Microflora in Newborns

“…Ранее изучение параметров колонизации кишечника бифидо-бактериями проводилось с использованием только куль-туральных методов, однако они не позволяют в полной мере оценить их видовой состав. В настоящее время широко применяют методы, основанные на изучении и сравнении генов рибосомальной рибонуклеиновой кис-лоты прокариот с коэффициентом седиментации 16 еди-ниц Сведберга (16S рРНК) [8].…”