2. Materials and Methods
2.1 Ethics statement
Blood samples were collected from animals in public slaughterhouses during the mandatory ante-mortem clinical examination. All procedures performed in this study followed common good clinical practices and received institutional approval from the Ethical Animal Care and Use Committee of the University of Naples Federico II (PG/2017/ 0099607). All farmers were previously informed and agreed with the purpose and methods used.
2.2 Blood samples and DNA extraction
Blood samples from 168 goats were obtained from the jugular vein and collected in vacutainers containing EDTA. Among these goats, 118 lived in close contact with cattle on lands rich in bracken fern (Pteridium spp.), which they grazed on. Cattle who share pastures with goats tend to develop chronic enzootic hematuria, a clinical syndrome caused by papillomavirus-associated bladder tumors. The remaining 50 goats were from flocks living in closed pens. They were fed fern-free hay and had no contact with cattle. Total DNA was extracted using a DNeasy Blood & Tissue Kit (Qiagen, Wilmington, DE, USA), according to the manufacturer’s instructions.
2.3 Digital droplet polymerase chain reaction
The primers and probes for the detecting four BPV genotypes (BPV-1, -2, -13, and -14) were used as reported elsewhere (De Falco et al., 2020). A black hole quencher was used together with the fluorescent reporter dyes FAM and VIC. The ddPCR assay mixture contained 10 μL of 2×ddPCR Supermix for Probes (Bio-Rad, Munich, Germany), 900 nmol/L of each primer, 250 nmol/L of probes, 5 μL of the DNA sample (100 ng), and enough sterile distilled water to ensure a final volume of 20 μL. Droplets were generated using a QX100 droplet generator (Bio-Rad, Munich, Germany), according to the manufacturer’s instructions. The droplets (~40 μL) were transferred to a 96-well plate, which was then heat-sealed. The PCR amplification was performed in a T100 Touch thermal cycler (Bio-Rad, Munich, Germany) using the following conditions: 95°C for 10 min; 40 cycles of 94°C for 30 s, 56°C for 1 min, and 98°C for 10 min; final hold at 12°C. Next, the droplets were analyzed using a QX100 droplet reader (Bio-Rad, Munich, Germany). Data were analyzed using QuantaSoft software 1.4 (Bio-Rad, Munich, Germany). All tests were repeated twice. Manual thresholds were applied to both the BPV genotypes and the positive controls, according to our previous research (De Falco et al., 2020). In each run, a BPV-negative sample and a non-template control were included. The BPV concentration was 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 (20 μL) and dividing the result by the volume of DNA sample added to the reaction mixture at the beginning of the assay (5 μL).
2.4 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 final PCR volume was 20 μL containing: 1x TaqMan Universal Master Mix (Applied Biosystems), 900 nM each of the forward and reverse primers, 250 nM of the probe, and 100 ng of the DNA sample. The following thermal cycling program was used: 50°C for 2 min; 95°C for 10 min; 40 cycles of 95°C for 15 s and 58°C for 60 s. Each sample was analyzed in duplicate, and the Ct values were determined using regression analysis. Data acquisition and data analyses were performed using CFX MaestroTM(Bio-Rad) software. The same samples used as positive controls for ddPCR were also tested using real time qPCR.
2.5 Statistical analysis
Differences in the proportions of cases detected were tested using the χ2 test of Campbell and Richardson (Richardson, 2011), with p-values ≤ .05 indicating statistical significance.