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.