Quantitative Microbiome Analysis by qPCR

Quantitative microbiological analysis of samples was carried out in qPCR experiments analyzed using SYBR® green methodology in a ViiA7 Real-Time PCR System (Life Technologies, Carlsbad, CA, USA). Primers, amplicon size, and annealing temperature for Akkermansia, Bacteroides, Bifidobacterium, Enterobacteriaceae, Faecalibacterium, Lactobacillus, Enterococcus, Prevotella, Roseburia, Blautia coccoides-Eubacterium rectale Cluster XIVa, Ruminococcus Cluster IV, and Clostridium leptum subgroup specific cluster IV have been described previously^{40 (link)}. For the analysis of B. fragilis and Bilophila we used the primers and PCR conditions described by Sjögren et al.^{41 (link)} and Baldwin et al.^{42 (link)}, respectively. DNA from E. coli DH5α, L. plantarum IFPL935, Enterococcus faecalis IFPL 382, Bifidobacterium breve 29M2, and B. fragilis DSM2151 were used to quantify total bacteria and Enterobacteriaceae, Lactobacillus, Enterococcus, Bifidobacterium, and Bacteroides and B. fragilis, respectively. For all other groups analyzed, samples were quantified using standards derived from targeted cloned genes using the pGEM-T cloning vector system kit (Promega, Madison, Wisconsin, USA) as described previously³⁷ .