DNA was extracted from the primary and secondary enrichment broths of the animal and meat samples using the boiling method described previously by de Medici et al. (2003) [28] (link). Five microliters of DNA template extracted was used in the real-time iQ™ Multiplex Powermix (Bio-Rad Laboratories, Hercules, CA, USA), in a final volume of 20 µL per reaction.
The real-time PCR assay targeted the following genes: nuc (identification of S. aureus), mecA (associated with methicillin resistance) and PVL-encoding genes (virulence factor) (Table 1 ).
The final concentrations in the reaction mixture were: 300 nM of primers (forward and reverse), 200 nM of fluorogenic probes (Applied Biosystems, Foster City, CA, USA), and 1X iQ™ Multiplex Powermix (Bio-Rad Laboratories, Hercules, CA, USA), according to the manufacturer's recommendations.
The thermal cycling conditions were adjusted to an initial denaturation of 3 min at 95°C, followed by 40 PCR cycles of 95°C for 15 s and 55°C for 1 min, using an iCycler IQ™ real time PCR system (Bio-Rad Laboratories, Hercules, CA, USA). An external positive control (DNA extracted from MRSA ATCC 35591, positive for mecA and PVL genes) and an external negative control (DNase/RNase-free distilled water) were included with each plate. Data analysis was carried out using the iCycler software version 3.0 (Bio-Rad Laboratories, Hercules, CA, USA).
The real-time PCR assay targeted the following genes: nuc (identification of S. aureus), mecA (associated with methicillin resistance) and PVL-encoding genes (virulence factor) (
The final concentrations in the reaction mixture were: 300 nM of primers (forward and reverse), 200 nM of fluorogenic probes (Applied Biosystems, Foster City, CA, USA), and 1X iQ™ Multiplex Powermix (Bio-Rad Laboratories, Hercules, CA, USA), according to the manufacturer's recommendations.
The thermal cycling conditions were adjusted to an initial denaturation of 3 min at 95°C, followed by 40 PCR cycles of 95°C for 15 s and 55°C for 1 min, using an iCycler IQ™ real time PCR system (Bio-Rad Laboratories, Hercules, CA, USA). An external positive control (DNA extracted from MRSA ATCC 35591, positive for mecA and PVL genes) and an external negative control (DNase/RNase-free distilled water) were included with each plate. Data analysis was carried out using the iCycler software version 3.0 (Bio-Rad Laboratories, Hercules, CA, USA).