Optimizing the primer and probe experimental conditions for qRT-PCR and digital PCR

Next, to set the optimal experimental conditions of primers and probes for application to qRT-PCR and digital PCR, preferentially, we performed the qRT-PCR validation using PC1 template DNA sample with S. aureus–specific primer, and the primer concentration was set the 25 pmol, probe concentration was set 20 pmol, and 10 pmol conditions, respectively. The qRT-PCR (CFX Opus 96 Real-Time PCE System, Bio-Rad; BioFACT 2× Multi-Star Real-Time PCR Master Mix For Probe, UDG system) experimental conditions were as follows (Supplementary Table 2); pre-denaturation 95.0°C 10 min, denaturation 95.0°C 10 s, annealing/Flour detection 61°C 10 s, elongation 72.0°C 15 s, and total PCR cycle was 45). As a result, the resolution of detection fluorescence value (RFU; relative fluorescence units) for the S. aureus greA gene detected on the probe concentration condition of 20 pmol about PC1 and PC2 samples were found to be higher compared to the 10 pmol concentration condition, confirming that this experimental condition was the most optimal (Fig. 1B).