Identification of LA-MRSA with mecC Gene

Livestock associated Methicillin resistant S. aureus harboring mecC gene were identified by conducting PCR on all phenotypically identified MRSA isolates with positive growth on MRSA selective agar, ORSAB. Specific primers for mecC genes (Table 1) as described earlier [21 (link)] were used to identify mecC positive LA-MRSA isolates. Two microliters of sample were added to 48 µL of master mix containing 26.5 µL nuclease free water, 10 µL 5X buffer, 2 µL 50 mM MgCl2, 1 µL 10 mM dNTPs, 3.75 µL for both 10 µM mecC R and mecC F and 1 µL Taq DNA polymerase (5 u/µL). The PCR protocol was set as pre-denaturation at 95 °C for 2 min, 30 cycles of amplification with denaturation at 95 °C for 45 s, annealing at 55 °C for 1 min, extension at 72 °C for 2 min and final extension at 72 °C for 5 min. The PCR products were analyzed by gel electrophoresis using 1.2% agarose and gel imaging was done using Gel Doc^TM EZ Imager (Bio-Rad, Hercules, CA, USA). The expected amplification product of 304 bp signifies a positive detection of mecC gene.