16S qRT-PCR for 16S and beta-actin genes was done to compare the host to microbial DNA ratio across the different sampling methods.

The primers used to amplify the 16S rRNA gene were: forward primer 341F 5′-CCTACGGGAGGCAGCAG-3′, and reverse primer 534R 5′-ATTACCGCGGCTGCTGGCA-3′. These primers are complementary to the conserved regions in the 16S rRNA gene, nucleotide positions 290–484 in Escherichia coli, producing a fragment of 195 bp.

The primers used to amplify the beta-actin Sus scrofa gene were: Forward primer 5′-CCAAGCCTGGACTACCTCCT-3′, and reverse primer 5′-AAACCTGGAGAGGTTCACCG-3′. These primers were complementary to the Sus scrofa actin beta (ACTB) transcript RNA gene, spanning the nucleotide positions 1,348–1,535, producing an amplicon length of 188 bp.

Primers were synthesized by Eurofins Genomics (Ebersberg, Germany). The final PCR mix contained 0.4 μl each of forward and reverse primers (total concentration of 0.4 nmol each), 5 μl of SYBR Green Master Mix (TaKaRa BioTech Corporation, Dalian, China), 3.2 μl of UltraPure DNase/RNase-Free distilled water (ThermoFisher Scientific, Waltham, MA, USA) and 1 μl of unamplified genomic DNA, giving a final reaction volume of 10 μl. All samples were performed in triplicate. qPCR was performed using the Roche LightCycler®480 Instrument (Roche Diagnostics, Meyland, France) on the following programme: 1 cycle of 30 s at 95 °C, 40 cycles of 10 s at 95 °C, 30 s at 59 °C and 20 s at 78 °C.

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