Quantification of 16S rRNA gene by qPCR

To assist in identifying contaminating sequences, the 16S rRNA gene quantity in the diluted DNA templates used for the amplicon PCR was measured by qPCR. The qPCR assays were performed using a universal primer set (forward, 5′-CCA TGA AGT CGG AAT CGC TAG-3′; reverse, 5′-GCT TGA CGG GCG GTG T-3′) that has been used for bacterial DNA quantification in previous studies [114, 115]. The assays were carried out using the LightCycler 96 (Roche Applied Science, Basel, Switzerland) in a 10 μL reaction volume, which contained 2 μL of PCR-grade water, 1 μL diluted DNA template, 5 μL LightCycler 480 SYBR Green I Master Mix (Roche Applied Science) and 1 μL (3 μM) of each primer. Samples, together with the extraction blanks and mock, were run in duplicate in addition to Femto™ bacterial DNA standards (Zymo Research; catalog no., E2006) and a no-template control of the qPCR assay. The qPCR program encompassed an initial enzyme activation step at 95 °C for 2 min, 45 three-step cycles of 95 °C for 10 s, 60 °C for 30 s and 72 °C for 15 s, and a melting curve analysis at the end. Quantification cycle (Cq) values were determined using the second derivative method [116]. The specificity of qPCR amplification was confirmed by evaluating the melting curve of qPCR products and the band pattern on the agarose gel after electrophoresis. The inter-plate calibration factor was calculated following the method described in [117], using the bacterial DNA standards as inter-plate calibrators.