2. Materials and Methods
2.1 Ethics statement
Blood samples were collected from animals in public slaughterhouses during mandatory ante-mortem clinical examinations. The study was approved by the Ethical Animal Care and Use Committee of the University of Naples Federico II, Department of Veterinary Medicine and Animal Production, Naples, Italy (PG/2017/0099607). All procedures were performed in accordance with the relevant rules and regulations on animal welfare.
2.2 Liquid biopsy samples and DNA extraction
Blood samples from 165 healthy 1- to 3-year-old sheep were collected from the jugular vein in vacutainers containing ethylenediaminetetraacetic acid (EDTA). Total DNA was extracted using a DNeasy Blood & Tissue Kit (Qiagen, Wilmington, DE, USA), according to the manufacturer’s instructions.
2.3 Positive Controls
The positive controls of OaPV1 and OaPV 2 were artificially created plasmids (vector: pUCIDT-AMP), containing 270 and 603 base pairs of the sequence of E5 and the major capsid protein, respectively (IDT, Integrated DNA Technologies, IA, USA). The positive control of OaPV3 was a plasmid (vector: pUC19) that contained the complete genome of OaPV3, and the positive control tissue for OaPV4 was a cutaneous fibropapillomatosis sample, both from the Department of Veterinary Medicine of Sassari University (kind gifts from Prof. A. Alberti).
2.4 qPCR
Using the real-time qPCR assay, the online web interface from IDT (https://eu.idtdna.com/scitools/Applications/RealTimePCR/) primers and probes were designed. The amplicon length was set by the program to obtain 70 -150 bp within the target regions. The primers and probes used for the detection of the four OaPV genotypes (OaPV1-2-3 and 4) are reported in Table 1. Primers and probes were ordered as a mix with a primer-to-probe ratio of 3.6. The qPCR reaction mixture was prepared by adding 7 μL of template (100 ng genomic DNA), 10 μL of 2X SsoAdvanced™ Universal Probes Supermix (Bio-Rad Laboratories, Hercules, CA, USA), 1 μL of target probe (FAM) /primer mix (final concentration of 900 nM of each primer and 250 nM of probe) in a total volume of 20 μl. DNA quality and concentration were assessed using a Nanodrop (Thermo Scientific, MA, USA). Four separate PCR reactions were performed using the CFX96 Real-Time System of the C1000 TouchTMThermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA). 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 analysis were performed using the CFX MaestroTM (Bio-Rad Laboratories, Hercules, CA, USA) software. The same samples used as positive controls for ddPCR were also tested using qPCR.
2.5 ddPCR
For ddPCR, Bio-Rad QX100 ddPCR System was used according to the manufacturer’s instructions. The reaction was performed in a final volume of 22 μL containing 11 μL of ddPCR Supermix for Probes (2X; Bio-Rad), 0.9 μM primer, and 0.25 μM probe (Table 1) with 7 μL sample DNA corresponding to 100 ng. A black hole quencher was used in combination with FAM fluorescent dye reporters (Bio-Rad Laboratories, Hercules, CA, USA). The ddPCR mixture was placed into a 96-well PCR plate, and 7 µL of each sample was added to each well (Bio-Rad Laboratories, Hercules, CA, USA). The plate was transferred to an automated droplet generator (AutoDG, Bio-Rad Laboratories, Hercules, CA, USA). The AutoDG added 70 µL of droplet generation oil for the probe in every well, and each sample was partitioned into ~ 20,000 stable nano-droplets. The droplet emulsion (40 µL) was transferred into a new 96 well PCR plate and, then coated with a pierceable film heat sealed using a PX1 PCR Plate Sealer (Bio-Rad Laboratories, Hercules, CA, USA). PCR amplification was performed on a T100 Thermal Cycler (Bio-Rad Laboratories) 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. A 96-well PCR plate was placed on the reader. Data were analyzed using the QuantaSoft analysis tool (Bio-Rad Laboratories, Hercules, CA, USA). Poisson statistics were used to calculate the absolute concentration of OaPV DNA in each sample (Pinheiro et al., 2012). To discriminate between positive (blue) and negative (gray) droplets, a manual threshold line was used. There were also differences in the fluorescence amplitude range of the background (negative) droplets among the OaPV samples, that is, 4,000-8,000 for OaPV1; 3,000-6,000 for OaPV2; 4,000-10,000 for OaPV3; and 4,000-12,000 for OaPV4. 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 DNA sample added to the reaction mixture (7 µL) at the beginning of the assay. Each sample was analyzed in duplicate. Samples with very few positive droplets were re-analyzed to ensure that these low copy number samples were not due to cross-contamination.
2.6 Limit of detection (LoD) determination
The four OaPV viral genes were detected using qPCR and ddPCR standard curves of the positive controls used in serial dilutions. A calibration curve of the positive sample dilutions (log10) was plotted against the PCR cycles. The linear range was determined by diluting the positive controls from 105 to 10-1 copies/µL, detecting each dilution three times, taking the average value, and correlating the result with the theoretical value. In qPCR, the correlation of  R 2 more than 0.98 was similar with the requirements of the test, and a Ct value of 40 was set as the minimum amount of viral detection assay. The lower detection limit obtained by ddPCR with values <1 copies/µL indicated high sensitivity.
2.8 Statistical analysis
McNemar’s Test for Two Related Binomial Proportions (Conditional) was used to evaluate the agreement between the two tests performed on the same animals. To evaluate the difference of the four types of papillomavirus in the same animals, the Cochran-Armitage Test was performed. P-value < 0.05 was considered to be statistically significant.