3. Quantification of bacterial amounts by quantitative real-time PCR

The relative amounts of total bacteria were measured using quantitative real-time PCR based on the 16S rRNA gene. The 16S rRNA gene was amplified using the primers 340F (5′-TCCTACGGGAGGCAGCAG-3′) and 518R (5′-ATTACCGCGGCTGCTGG-3′) with the TaKaRa PCR Thermal Cycler Dice Real Time System III (Takara Bio Inc., Kusatsu, Japan). Triplicate reactions were performed for each sample with a final volume of 25 μL comprising 12.5 μL of 2X SYBR Premix Ex Taq (Takara Bio Inc.), 10 μM of each primer, and 2 μL of DNA template (ten-fold diluted metagenomic DNA) or distilled water (negative control). The conditions for the reaction were as follows: initial denaturation at 95°C for 30 seconds; 40 cycles of denaturation at 95°C for 5 seconds, annealing at 60°C for 30 seconds. Standard curves were generated from parallel PCRs with serial log-concentrations (1 × 10²-1 × 10⁸) of the copy number of the 16S rRNA gene from Escherichia coli w3110.