qPCR was carried out for quantification of 16S rRNA gene copy number to compare the bacterial load collected by each technique. qPCR was performed with universal BactQUANT 16S rRNA gene primers (Forward primer:5′-CTACGGGAGGCAGCA, Reverse primer: 5′-GGACTACCGGGTATCTAATC) (Sigma) with the FAM labelled BactQUANT probe ((6FAM) 5′-CAGCAGCCGCGGTA-3′ (MGBNFQ)) (Liu et al., 2012 (link); Mitra et al., 2017 (link)) on a CFX Real Time PCR system (Bio-Rad). A tenfold standard curve (3030 to 303,039,700 copies) of Escherichia coli genomic DNA (Sigma, D4889) was generated, each reaction contained 5 µL of DNA sample or standard, 10 µL Platinum PCR Super mix UDG containing Rox (Life Tech, 11730-017) and primers and probe. Thermal cycling was performed at 3 min at 50°C for UNG incubation,10 min at 95°C for Taq activation, then 40 cycles of 15 s at 95°C for denaturation and 1 min at 60°C for annealing and extension. Cycle threshold (Ct) value for each reaction were obtained using CFX Maestro software version 1.1 (Bio-Rad) after application of fluorescence drift correction, background subtraction using the ‘curve fit’ option and automatic Ct baseline definition. Ct values were converted to 16S rRNA gene copy number from the generated standard curve. Samples were run in duplicate; a negative control of molecular grade water was included to eliminate contamination.
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