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

Abstract

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.

Simulating colonic survival of probiotics in single-strain products compared to multi-strain products

“…The SYBR Green methodology (Applied Biosystems) was used for B. lactis Bi-07, B. lactis Bl-04, Lactobacillus spp. [10] and L. acidophilus , while the TaqMan methodology was used for Bifidobacterium spp. [9], L. acidophilus and L. paracasei [11].…”

Safety ofBifidobacterium animalissubsp.lactis(B. lactis) strain BB-12-supplemented yogurt in healthy adults on antibiotics: a phase I safety study

“…DNA concentration was determined by NanoDrop (Thermo Fisher Scientific, Waltham, MA). The amount of bacterial copy numbers in fecal DNA was determined by real-time PCR using previously validated primers and probe sets to identify a common 16S rRNA (rRNA) sequence fragment of Domain bacteria for determination of total bacterial load, 25 Lactobacillus and Bifidobacterium species, [26][27][28] Bacteroides fragilis group (B. fragilis group) 29 or using a specific primer-probe set for identification of a genomic tuf gene fraction used for identification and relative quantification of Bifidobacterium animalis subsp lactis. 24 A bacterial strain representative for each bacterial species was used as a positive amplification control.…”

Effects of feeding finisher pigs with chicory or lupine feed for one week or two weeks before slaughter with respect to levels of Bifidobacteria and Campylobacter

“…and total bacteria in the luminal content from the distal ileum and the caecum were measured according to published qPCR assays. The gene sequences of the primers for bifidobacteria were F-bifido 5 0 -CGCGTCYGGTGTGAAAG-3 0 and R-bifido 5 0 -CCC CACATCCAGCATCCA-3 0 (Delroisse et al, 2008), and for total bacteria 8FM 5 0 -AGAGTTTGATCMTGGCTCAG-3 0 and Bact515R 5 0 -TTACCGCGGCKGCTGGCAC-3 0 (Palmer et al, 2007). The probes MGB-bifido (5 0 AACAGGATTAGATACCC-3 0 ) and Bact338K (5 0 -CCAKACTCCTACGGGAGGCAGCAG-3 0 ) were labelled at their 5 0 end with the reporter dye 6-carboxyfluorescein (FAM).…”

“…Quenchers at the 3 0 end were a non-fluorescent quencher dye (bifido) and 6-carboxytetramethylrhodamine (TAMRA) (total bacteria). The qPCR was performed on RotorGene 3000 (Corbett Research, Mortlake, New South Wales, Australia) with PCR conditions as described by Delroisse et al (2008) and Palmer et al (2007). For each analysis, 2 ml of total DNA, diluted 1 : 10 to minimize inhibitory effects, was used as the template.…”