qPCR quantification of fecal bacterial groups

The fecal microbiota from KTRs was explored at various taxonomic levels through the quantification of nine bacterial groups or species by qPCR: The Firmicutes/Bacteroidetes ratio, Bacteroides-Prevotella group, Lactobacilli, Bifidobacteria, Akkermansia muciniphila, Faecalibacterium prausnitzii, Escherichia coli, Clostridium coccoides, and Clostridium leptum.

All these bacteria or bacterial groups were specifically chosen as they have been shown to be associated with metabolic disorders in mice and/or the population of non-transplanted patients.

After extraction, fecal bacterial DNA was quantified using a Nanodrop^® analyzer and diluted in order to obtain a concentration of 10 ng/μl. qPCRs included the template (50 ng of DNA per reaction), 5 μL of 2X SYBR Green mix (Absolute blue^® qPCR SYBR Green, Thermo scientific^®, including Taq hot start enzyme) and each primer to a final concentration in the mix of 300 nM. Water was added to obtain a final reaction volume of 10 μL. The sequence, and annealing temperature of primer pairs used to quantify each bacterium or bacterial group is shown in S1 Table. qPCRs were carried out in a LightCycler480 (Roche^®) as follows: one initial activation step of 15 min at 95°C, 40 cycles of 2-step amplifications (95°C for 15” for denaturation, 57–63°C for 1 min for annealing). A bacterium or a bacterial group was considered undetectable in a sample if its quantification cycle (Cq) was ≥ 35.

All qPCRs were followed by a dissociation curve to check for the amplification of a unique DNA fragment.

All bacterial quantifications were performed on two independent qPCRs, each containing a duplicate of each sample in the same 96 well plate, with the result calculated as the mean of the 4 measures. Template DNA was thawed only twice, once for each repeat of the qPCR.

The relative amount of a bacterium or a bacterial group in a given sample was inferred with the following formula:

where Q_Bac is the (log-) relative abundance of the studied bacterium or bacterial group in the sample. The formula is based on the qPCR cycle number (Cq) where the SYBR Green signals exceed the detection threshold: CqEub is the mean Cq obtained with the “Eubacteria” pair of primers (see S1 Table) which quantifies all bacteria in the sample, and CqBac is the mean Cq obtained with the pair of primers specific of the studied bacterium or bacterial group.