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.