Molecular Detection of MRSA Isolates

Definite identification of MRSA isolates was dependent on the detection of their characteristic mecA gene [15 (link)]. Briefly, genomic DNA was extracted using GeneJET Genomic DNA Purification Kit according to the manufacturer’s instructions (Thermo Fisher Scientific Inc., Vilnius, Lithuania). The mecA gene was amplified, by PCR technique, using a Thermal cycler (Biometra UNO–Thermoblock, Germany) in presence of 1 µL from each particular 10 pmol primer (Table 1), 12.5 µL Cosmo PCR Red Master Mix (Willowfort, Birmingham, UK), 1 µL of 50 ng genomic DNA, and nuclease-free double distilled water to final reaction volume of 25 µL. The mecA gene amplification cycling program includes 1 cycle of initial denaturation at 94 °C for 4 min (min) followed by 35 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, and elongation at 72 °C for 1 min, and lastly 1 cycle of final elongation at 72 °C for 10 min. The obtained PCR products were loaded into 1.5% agarose gel containing 5 µL ethidium bromide (0.05 mg/mL) followed by electrophoresis at 100 V in Tris-acetate EDTA buffer (Sigma Aldrich, Hamburg, Germany). The UV transilluminator (HERMLE Labortechnik GmbH, Wehingen, Germany) was used to observe the mecA gene-specific bands and determine their molecular size with the aid of a 100 bp DNA ladder (Geneaid Biotech Lt., New Taipei City, Taiwan) as a marker [16 (link)].