One-Step reverse transcription (RT)-ddPCR
Total RNA was extracted from 34 healthy cows (as negative controls) as previously reported. 100 ng of total RNA was used for One-Step RT-ddPCR Advanced Kit for Probes (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions. The reaction was performed in a final volume of 22 μL, containing 11 μL of ddPCR Supermix 2x for Probes, 1 μL of primer and probe mix for OaPVs (Table 1), 2 μL reverse transcriptase, and 1 μL DTT. The plate was transferred to an automated droplet generator (AutoDG, Bio-Rad Laboratories, Hercules, CA, USA) as described above. PCR amplification was carried out on a T100 Thermal Cycler (Bio-Rad Laboratories Hercules, CA, USA) with the following thermal profile: 50°C for 60min, 95°C for 10 min, 40 cycles of 94°C for 30 s, 58°C for 1 min, 1 cycle at 98°C for 10 min, and ending at 4°C. After amplification, the plate was loaded onto a droplet reader (Bio-Rad Laboratories, Hercules, CA, USA), and the droplets from each well of the plate were read automatically. Therefore, the ddPCR results could be directly converted into copies/µL in the initial samples simply by multiplying them by the total volume of the reaction mixture (22 µL) and then dividing the number by the volume of the RNA sample added to the reaction mixture (5 µL) at the beginning of the assay. Each sample was analyzed in duplicate.