Quantification of 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., Escherichia coli, and Lactobacillus were cloned into the pMD19-T vector (21 (link)). Gene sequences were amplified from fecal total DNA using the primers listed in Table 2. A total of five 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 of 10²–10¹⁰ DNA copies/mL using a Lightcycler 480II instrument (Applied Biosystems, Carlsbad, CA, USA). The general microbial DNA extracted from feces and specific DNA from recombinant microbiota were quantified by RT-PCR. Reaction conditions were 2 min at 50°C, an initial denaturation step at 95°C for 5 min, and then 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 (22 (link)).