Fluorogenic probes

Genomic DNA was extracted in a QIAcube instrument following the manufacturer’s standard protocol for saliva nucleic acid extraction (QIAGEN, Valencia, CA). After isolation, allelic discrimination for the ADA gene was determined via real-time polymerase chain reaction (PCR) using a TaqMan SNP genotyping assay using fluorogenic probes (Applied Biosystems, CA). Thermal cycling was performed on StepOne Real-Time PCR system (Applied Biosystems, CA). The amplification mix contained the following ingredients: 12.5 μL of PCR master mix (QIAGEN, Valencia, CA), 1.25 μL of TaqMan 20X working stock, 10.25 μL of RNase- and DNase-free water (Sigma), and 1.0 μL of sample DNA, in a total volume of 25 μL per single tube reaction. The PCR conditions were 95 °C for 10 minutes followed by 40 repeated cycles of 95 °C for 15 seconds and 60 °C for 60 seconds. Genotypes were determined automatically via the StepOne software (Applied Biosystems, CA) based on the fluorescence signals. Samples were run in duplicate and in the case of a call discrepancy, samples were rerun.

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).

Genomic DNA was extracted in a QIAcube instrument following the manufacturer's standard protocol for saliva nucleic acid extraction (QIAGEN, Valencia, CA, USA). After isolation, allelic discrimination for the COMT gene was determined via real‐time polymerase chain reaction (PCR) using a TaqMan SNP genotyping assay using fluorogenic probes (Applied Biosystems, CA, USA). Thermal cycling was performed on StepOne Real‐Time PCR system (Applied Biosystems). The amplification mix contained the following ingredients: 12.5 μl of PCR master mix (QIAGEN), 1.25 μl of TaqMan 20× working stock, 10.25 μl of RNase‐ and DNase‐free water (Sigma), and 1.0 μl of sample DNA, in a total volume of 25 μl per single‐tube reaction. The PCR conditions were 95°C for 10 min followed by 50 repeated cycles of 92°C for 15 s and 60°C for 90 s. Genotypes were determined automatically via the StepOne software (Applied Biosystems) based on the fluorescence signals. Samples were run in duplicate and in the case of a call discrepancy, samples were rerun.