The diagnostic value of the droplet digital PCR for the detection of bovine deltapapillomavirus in goats by liquid biopsy

Abstract

Papillomaviruses (PV) are small DNA viruses that have co-evolved for millions of years with various species, including mammals, reptiles and birds (van Doorslaer, 2013). As of now, over 300 PV genotypes have been discovered. Bovine papillomaviruses (BPVs) are the second most studied PVs beyond human papillomaviruses (HPVs) (Daudt et al., 2018). BPVs comprise 29 genotypes classified in five genera (http://pave.niaid.nih.gov/; Yamashita- Kawanishi, Ito, et al., 2020;. The deltapapillomavirus genus (δPVs) is composed of four highly pathogenic BPVs, namely BPV-1, BPV-2, BPV-13 and BPV-14 (Daudt et al., 2018).Bovine BPVs recognize Bos taurus as their classical host. Indeed, BPVs play an important aetiological role in both cutaneous and mucosal tumours of cattle. BPV-2 and BPV-13 are the most important infectious agents associated with bladder tumours as they are found very commonly in some breeds of pasture-residing cattle that graze on bracken fern-infested lands (Campo et al., 1992;Roperto et al., 2013Roperto et al., , 2016. However, BPVs are also the only papillomaviruses capable of natural cross-species transmission and infection

Detection and quantification of bovine papillomavirus DNA by digital droplet PCR in sheep blood

“…To our knowledge, this study is the first to use the sensitive ddPCR assay, a biotechnological refinement of conventional PCR, to detect and quantify highly pathogenic bovine δPVs in clinically healthy sheep by liquid biopsy. Our previous studies have shown that ddPCR has both high specificity and sensitivity for the detection and quantification of BPV DNA in healthy and diseased cattle 7 as well as in healthy goats 19 . The current study provides further evidence that ddPCR is a very useful approach to detect and quantify BPV in the blood of healthy sheep and allows us to gain diagnostic and epidemiological insights into BPV presence in ovine species as data on the prevalence and types of BPVs in sheep are not currently available.…”

“…Four separated PCR reactions were performed on the CFX96 Real-Time System of the C1000 Touch Thermal Cycler (Bio-Rad Laboratories) 7 , 19 . 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.…”

“…Quantification of PVs by digital PCR is proving to be a valuable improvement over qPCR, as it has been shown to have a higher robustness to mismatches between the primer-probe set and PV genotypes 7 , 19 . Due to pathogens that cause latent infection, BPV concentrations in the blood are sometimes too low to be determined by traditional methods.…”

“…Due to pathogens that cause latent infection, BPV concentrations in the blood are sometimes too low to be determined by traditional methods. In cattle and goats, ddPCR has been found to outperform qPCR in terms of the sensitivity, specificity, and reproducibility of BPV detection, all of which play a central role in diagnostic and epidemiological procedures to identify the geolocalization of BPVs 7 , 19 .…”

Droplet Digital PCR (ddPCR) Analysis for the Detection and Quantification of Cow DNA in Buffalo Mozzarella Cheese

“…Unlike real-time PCR (qPCR), this method yields absolute concentrations without requiring the presence of a standard calibration curve, thus making the process faster, more precise, and reproducible [ 13 ]. Furthermore, the ddPCR is used for several applications such as the detection of viruses, bacteria, and parasites involved in different diseases [ 14 , 15 ].…”