16S rRNA gene amplicon of meat production microbiota: Pork Carcass opening
Swabs (~1400cm2 area) were taken along the meat exposed opening of the carcass from the hind leg to the oral cavity, using sterile gauze pads (10x10 cm, 16 ply) moistened with 15 ml 0.9% saline, and homogenized in stomacher bags with 25 ml 0.9% saline. 10 ml swab homogenate was centrifuged for 20 min at 2,880 x g, and the pellets resuspended in 1 ml 0.9% saline. DNA extracted using MoBio PowerLyzer® Power Soil® DNA Isolation Kit modified by using a Mixer Mill MM 300 for 10 min at 30 cycles per second. DNA was diluted ten-fold and Two-step PCR of the v3-v4 region was performed, First 30 cycles with broad range primers (341F and 806R), then 15 cycles to add sequencing primers and adaptors. 250 bp Paired-end sequencing were performed on the Illumina MiSeq System using MiSeq Reagent Kits v2.