Gut Microbiome Profiling by qPCR

DNA was extracted from fresh faecal samples immediately after subsampling using the Norgen Stool DNA Isolation Kit (Norgen Biotek Corp., Thorold, ON, Canada), according to the manufacturer’s instructions, including a bead beating step. DNA purity and concentration were measured using a DS-11 DeNovix spectrophotometer (DeNovix Inc., Wilmington, DE, USA). Purified DNA was diluted to 50 ng/µL and stored at −20 °C until used for qPCR amplification. Quantification of Firmicutes and Bacteroidetes [24 (link)], Clostridium cluster I [25 (link)], Lactobacillus spp. [26 (link)], Bifidobacterium spp. and Enterococcus spp. [27 (link)] were evaluated by qPCR using specific oligonucleotides (Table S2). SYBR-based qPCR assays were performed following the run protocol described previously [23 (link),28 (link)]. Each sample was run in duplicate while standard curves were run in triplicate.