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.