4.4. DNA Extraction and Molecular Identification of S. aureus

DNA from all presumptive S. aureus isolates was extracted using the conventional boiling method as previously described [57]. All extracted DNA was stored at −20 °C until used for polymerase chain reaction (PCR) amplification. S. aureus was confirmed by amplifying a housekeeping gene nuc. S. aureus was identified as MRSA by amplifying mecA and mecC genes. PCR was performed in a 25 μL reaction tube with 1 μL template DNA, 2.5 μL 10×rTaq Buffer (Mg²⁺Plus) (TaKaRa company, Dalian, China), 2 μL dNTP Mixture (2.5 mM) (TaKaRa company, Dalian, China), 0.125 μL TaKaRa Taq (5 U/μL) (TaKaRa company, Dalian, China), 0.5 μL of each forward and reverse primers, and 18.375 μL ddH₂O. The following PCR conditions were used; 5 min at 94 °C; 30 cycles of 30 s at 94 °C, 1 min at annealing temperature, and 1 min at 72 °C, and final extension at 72 °C for 10 min. Each PCR was conducted in triplicate using a thermocycler (BioRad, Hercules, CA, USA). PCR products were visualized on 1% agarose gel, stained with ethidium bromide, using electrophoresis for 10 min at 175 volts with 0.5×TBE, and visualized under UV light using the Bio ChemiDoc imaging system (BioRad, California, USA). The nuc, mecA, and mecC genes primers and their positive controls are shown in Supplementary Table S1. DNase free water was used as negative controls.