Profiling Gut Microbiome Composition

Total microbial DNA was extracted and purified using a QIAamp DNA Stool Kit (Qiagen, Hilden, Germany) and stored at -80°C. The 16S rRNA gene sequences of Bacteroidetes, Bifidobacterium spp., Clostridium cluster IV, Clostridium cluster XIVa, Escherichia coli, Firmicutes, Lactobacillus, and total bacteria (Raveh-Sadka et al., 2015 (link)) were cloned into the pMD19-T vector. Gene sequences were amplified from total colonic DNA using the primers listed in Table 3. A total of eight clones with 16S rRNA gene sequences belonging to different taxa were used as templates to test primer specificity. Standard curves were constructed with DNA from representative species for a concentration range from 10² to 10¹⁰ DNA copies/mL using a Lightcycler 480II instrument (Applied Biosystems). General microbial DNA extracted from colonic contents and specific DNA from recombinant microbiota were quantified using RT-PCR. The reaction conditions were as follows: 2 min at 50°C; an initial denaturation step at 95°C for 5 min; 40 cycles of denaturation at 94°C for 20 s, primer annealing at a species-specific temperature for 30 s, and primer extension at 60°C for 1 min (Decroos et al., 2006 (link)).