Confirmatory Colony PCR for S. aureus

To confirm the presence of S. aureus, we used colony PCR with femB primers. The primers were FemB1 (5′-CAT GGT TAC GAG CAT CAT GG) and FemB2 (5′-AAC GCC AGA AGC AAG GTT TA), leading to an S. aureus-specific 447-bp PCR product (65 (link)). We touched a pipette tip onto a single S. aureus colony and mixed it with 20 μL of water in a PCR tube. We boiled the samples for 5 min at 95°C. Each sample contained 2 μL of a boiled cell solution and 18 μL of the PCR master mix with MyTaq DNA polymerase (Bioline). The PCR cycling conditions were 95°C 5 min; 30 cycles of 95°C for 15 s, 58°C for 10 s, and 72°C for 30 s; and 72°C for 10 min. We added 2.5 μL Sybr Safe DNA gel stain (Thermo Fisher, UK) to 100 μL of an agarose solution, which contained 1% (wt/vol) agarose dissolved by heating in 1× TBE (Tris-borate-EDTA) buffer. We loaded 15 μL of the PCR mixture into each well, with a 100-bp 5-μL 5 HyperLadder (Bioline) to confirm the product size. Electrophoresis was performed in a Sigma-Aldrich electrophoresis tank with 1× TBE at 100 V for 40 min. Electrophoresed gels were visualized under blue light, and their images were obtained with the GelDoc XR system imager (Bio-Rad).