Quantification of Bifidobacterium spp. and Lactobacillus spp. in rat fecal samples by real-time PCR

The microbiota of the rat intestinal tract constitutes a complex ecosystem of microorganisms. We have developed a real-time quantitative PCR assay based on genus-specific 16S rDNA primers and 3' minor groove binder (MGB) probes for accurate detection and quantification of a wide range of Bifidobacterium spp. (30 species) and Lactobocillus spp. (15 species) in rat fecal samples. Real-time PCR detection of serially diluted DNA isolated from reference strains of Bifidobacterium longum and Lactobacillus acidophilus was linear for cell counts ranging from 10(6) to 10 cells per PCR assay. The method proved applicable to the detection of Bifidobacterium spp. and Lactobacillus spp. at concentrations down to 10 CFU per PCR, corresponding to 5 x 10(4) CFU/g feces. The inter-extract reproducibility was high, with a coefficient of variation ranging from 0.24% to 1.07% for the Bifidobacterium assay and from 0.05% to 1.28% for the Lactobacillus assay. We conclude that real-time PCR is a very sensitive and precise technique for extensive quantitative evaluation of gut Bifidobacterium spp. and Lactobacillus spp. Thus, the approach used here to detect and quantify bacteria with group-specific primers should contribute to further studies of the composition and dynamics of the rat intestinal microbiota.