2. Materials and Methods
2.1 Ethics statement
The authors confirm that the ethical policies of the journal, as noted on the journal’s Author Guidelines page, have been adhered to. No ethical approval was required for this study, as we did not perform any animal experiments. All blood samples were collected at public slaughterhouses. 2.2 Blood samples and DNA extraction Blood samples were harvested from 40 cows that had grazed on lands contaminated with bracken fern (Pteridium spp.) and collected into EDTA-containing anticoagulant tubes. Fifteen of these cows (3-18 years of age) had been suffering from chronic enzootic hematuria for several years. The other 25 cows (3-20 years of age) were healthy animals that had been in close contact with the diseased cows as they belonged to the same cattle breeding farm. These 25 cows were considered healthy as they did not exhibit any bladder lesions, which may be a clinical marker of BPV infection. The cows were slaughtered at public slaughterhouses located in three regions of southern Italy (Basilicata, Calabria, and Campania), according to the decision of the owners of the animals. Medical authorities had carried out an ante-mortem clinical examination of the animals according to national legislation. Total DNA was extracted using a DNeasy Blood & Tissue Kit (QiagenTM, Wilmington, DE, USA), according to the manufacturer’s instructions. 2.3 Digital droplet polymerase chain reaction
The simplex PCR designs were for four BPV genotypes (BPV-1, -2, -13, and -14). Table 1 reports the primers and probes used for the detection of these four genotypes as well as their GenBank accession numbers. A black hole quencher was used in combination with the fluorescent reporter dyes FAM and VIC. In brief, each PCR mixture (total volume 20 µL) contained 1x ddPCRTM Supermix for Probes (no dUTP; Bio-Rad, Mississaugua, ON, Canada) 0.9 μM of the primer and 0.25 μM of the probe (Applied Biosystems, Hilden, Germany) and 5 μL of the DNA sample (100 ng). The Mastermix and DNA sample were thoroughly mixed and then transferred to a DG8 cartridge for placement in the QX100™ Droplet Generator (Bio-Rad). Next, the Droplet Generation Oil for Probes (Bio-Rad) was added to the cartridge. After their generation, 40 µL of the droplets was carefully transferred to a twin.tecTMsemi-skirted 96-well PCR plate (Eppendorf AG, Hamburg, Germany). The plate was sealed twice (4 s at 170 °C each time) using an Axygen Platemax semi-automated plate sealer (Thermo Fisher, Scientific, Waltham, MA, USA). Then, amplification was carried out in a T100 96-well thermal cycler (Bio-Rad). Two thermal gradients ranging from 57 °C to 62°C were tested to determine the optimal hybridization temperature of the assay. The PCR cycling conditions were as follows: enzyme activation at 95 °C for 10 min; 40 cycles of denaturation at 94 °C for 30 s and 56 °C for 1 min; enzyme deactivation at 98 °C for 10 min; and a final hold at 12° C. The droplets were quantified in a QX100 Droplet Reader™ (Bio-Rad) after which the ddPCR data were analyzed using Quantasoft Version 1.4. Manual thresholds were applied to both the BPV genotypes and the positive controls, the latter of which were BPV-1 DNA from a zebra sarcoid (Romanucci et al, 2020) (a kind gift provided by Dr. Borzacchiello, University of Naples, Italy); BPV-2 clone DNA (a kind gift by Dr A. Venuti, IRCSS Regina Elena, National Cancer Institute, Rome, Italy); and BPV-13 and -14 DNA from bovine bladder tumors from our laboratories (Roperto et al., 2016a, b). In each run, a BPV-negative sample and a non-template control were included. The BPV concentration was finally expressed as the number of copies of DNA per microliter of blood (copies/μL). Therefore, the PCR result could be directly converted into copies/μL in the initial samples simply by multiplying it with the total volume of the reaction mixture (22 μL) and then dividing that number by the volume of DNA sample added to the reaction mixture (5 μL) at the beginning of the assay.
2.4 Optimization of the digital droplet polymerase chain reaction assay of papillomaviruses
The thermal gradient was tested in the range of 55-62 °C to determine the optimal hybridization temperature of the assay. The most suitable hybridization temperatures for the efficient detection of the bovine δPVs were 58 °C for BPV-1, -13, and -14 and 56 °C for BPV-2. At these temperatures, the positive droplets showed the highest fluorescence amplitude and were well distinguishable from the negative droplets. To optimize the assay, three different DNA volumes were tested: 1, 4, and 5 μL. The results showed that increasing the DNA volume added to the PCR mixture increased the number of DNA copies detected, without complete inhibition of the reaction. As the aim of the ddPCR assay in this study was also to investigate the limit of detection of δPVs in low-level contaminated samples, the final DNA volume chosen was 5 μL, which corresponded to the highest volume of DNA that could be added to the ddPCR mixture in this study. The threshold was manually set at the fluorescence amplitude for each ddPCR run.
2.5 Quantitative real-time polymerase chain reaction
The qPCR assays were performed on the CFX96 Real-Time System of the C1000 TouchTM Thermal Cycler (Bio-Rad), using 96-well plates (Hard-Shell® 96-Well PCR Plates, #hsp9601; Bio-Rad). The following thermal cycling program was used: 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. The reaction mixture (final volume 20 μL) contained 1x TaqMan Universal Master Mix (Applied Biosystems), 900 nM of each of the forward and reverse primers, 250 nM of the probe (Table 1), and 5 μL of the DNA sample. Each sample was amplified in duplicate, and three independent PCR runs were carried out to obtain nine Ct values for the sample. The data acquisition and data analyses were performed using CFX MaestroTM(Bio-Rad). The Ct values were determined using regression analysis, where a value of ≥ 38 was considered to be a negative result, according to the cutoff indicated by Harper et al. (2010).
2.6 Statistical analysis
Differences in the proportions of detected cases were tested using the χ2 test of Campbell and Richardson, with P ≤ 0.05 indicating statistical significance. All analyses were carried out using the R System. According to Damerla et al. (2019), the sensitivity of the assay was calculated as the number of blood samples positive for circulating BPV DNA divided by the total number of examined cows (N = 40).