ddPCR
Table 1 lists the primers and probes used for ddPCR. Primers and probes were obtained as a mixture containing a primer-to-probe ratio of 3.6 (final concentration of 900 nM of each primer and 250 nM of probe). For ddPCR, a Bio-Rad QX100 ddPCR system was used according to the manufacturer’s instructions. The reaction was performed in a final volume of 22 μL and contained 11 μL of ddPCR Supermix for Probes (2X; Bio-Rad Laboratories, Hercules, CA, USA), 1 μL of OaPVs primer and probe mixture, 7 μL of DNA samples (corresponding to 100 ng), and 3 μL of DNAase-free water. The plate containing the reactions was subsequently transferred to an automated droplet generator (AutoDG; Bio-Rad Laboratories, Hercules, CA, USA). AutoDG added 70 μL of droplet generation oil to each well, and each sample was partitioned into approximately 20,000 stable nanodroplets. The droplet generator transferred each row of 8 droplet emulsion (40 μL) champions into a new 96 well PCR plate, which was subsequently coated with a pierceable film heat-sealed using a PX1 PCR Plate Sealer (Bio-Rad Laboratories, Hercules, CA, USA). PCR amplification was performed using a T100 Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA) with the following thermal profile: hold at 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. The data were analyzed using the QuantaSoft analysis tool (Bio-Rad Laboratories, Hercules, CA, USA). Poisson statistics were used to calculate the absolute concentration of the OaPV DNA in each sample. A manual threshold line was used to discriminate between positive (blue) and negative (gray) droplets. There were also differences in the fluorescence amplitude range of the background (negative) droplets among the OaPV samples: 1,000–2,500 for OaPV1 E5, 3,500–7,000 for OaPV2 L1, 500–2,700 for OaPV3 E6, 2,000–7,000 for OaPV3 E7, and 1,000–4,000 for OaPV4 E6. 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 that number by the volume of the DNA sample added to the reaction mixture (7 μL) at the beginning of the assay. Each sample was analyzed in duplicate. According to previous studies on PV detection and quantification using ddPCR (De Falco et al., 2021a; 2021b; Jeannot et al., 2016; Jeannot et al., 2021), blood samples were considered OaPV-positive in the presence of at least three positive droplets at the same amplitude as positive controls. A sample was considered OaPV-negative when fewer than three droplets or no droplets containing OaPV amplicons were observed.