Forward and reverse primers

qPCR was performed in a final volume of 20 μL containing 10 μL of TaqMan Universal Master Mix (Applied Biosystems, Foster City, CA, USA), 900 nM of each of the forward and reverse primers (Bio-Rad Laboratories, Hercules, CA, USA), 250 nM of the probe (Bio-Rad Laboratories), and 100 ng of the DNA sample. The concentration of the DNA samples was determined by Nano Vue Plus (GE HealthCare, Boston, MA, USA). The following primers and probes were used for the detection of four BPV genotypes.
Four separated PCR reactions were performed on the CFX96 Real-Time System of the C1000 Touch Thermal Cycler (Bio-Rad Laboratories)^{7 (link),19 (link)}. The thermal cycling conditions were as follows: 50 °C for 2 min, 95 °C for 10 min, and 40 cycles of 95 °C for 15 s and 58 °C for 60 s. Each sample was analyzed in duplicate, and negative controls were included in all runs. Data acquisition and data analyses were performed using CFX Maestro (Bio-Rad Laboratories) software. The same samples used as positive controls for ddPCR were also tested using qPCR.

Total RNA was isolated using the TRIZOL Reagent (Invitrogen), quality and concentration of the RNA were determined by measuring A₂₆₀ with a spectrophotometer (Nanodrop ND-1000) and then converted to cDNA by using the iScript cDNA synthesis kit (BioRad #1708890). qPCR was performed by using SsoAdvanced Universal SYBER Green PCR master mix (BioRad # 1725274) using forward and reverse primers from BioRad and Eurofins to quantify mRNA level of gbp2, gbp5, ifi204, ligP, and the β2-microglobulin gene (b2m). The latter was amplified as an internal control. The unique primer sequences are listed in Table S1 and the ifi204 and ligP amplicons in Table S2. Amplification was performed by using QuantStudio5 with the following cycling parameters 95°C for 5 min, then 40 cycles of 95°C for 15 seconds, 60°C for 1 minute, and 95°C for 15 seconds, followed by a final extension at 60°C for 1 minute.
The Ct values of the selected genes were normalized using the b2m gene as a reference. The relative copy numbers were calculated using the equation RCN=2^-ΔCt (Gavrilin et al., 2006 (link)). ΔCt denotes the Ct of the indicated gene subtracted with the Ct of b2m.