2.4. Gut Microbiota Pattern Quantification by qPCR

The total microbial DNA was isolated from 1 mL of the culture using the PureLink Microbiome DNA purification kit (Invitrogen, USA). The concentration and purity of the DNA were measured with NanoDrop 8000 spectrophotometers (ThermoFisher Scientific, USA). Quantification of the number of genome copies of the principally significant microbial groups from among the human gut microbiota (Enterobacteriaceae family, Bacteroides–Prevotella–Porphyromonas group, Lactobacillus–Lactococcus–Pediococcus group, Firmicutes phylum, Bifidobacterium genus) was done by qPCR using the Applied Biosystem 7900HT Real Time-PCR system. Employing a pair of universal primers for the prokaryotes, the bacterial content in each sample was determined. Bacterial quantification was done by developing standard curves using serial dilutions of a known genomic DNA concentration corresponding to Escherichia (E.) coli ATCC 10536, Lactobacillus (L.) plantarum ATCC 8014, Bifidobacterium (B.) breve ATCC 15700, Enterococcus (E.) faecalis ATCC 29,212, and B. fragilis DSM2151. The mass of genomic DNA was converted in copy number of 16S rRNA gene according to the Applied Biosystems guide. The Power SyberGreen PCR Master Mix 2X (Applied Biosystems, USA), and 40 ng total DNA were introduced into a qPCR reaction with a 20 µL volume. The amplification conditions were 95 °C for 10 min followed by 40 cycles with 95 °C for 15 s and 60 °C for 60 s. Table 2 shows the sequence of the primers and prevailing conditions [14].

Primer sequences and conditions used in the qPCR reaction.