Droplet digital PCR (ddPCR) for enhanced detection and quantification of bovine viral diarrhea virus (BVDV): A promising tool for disease control.

Bovine Viral Diarrhea (BVD) significantly impacts cattle production and reproduction, causing substantial economic losses in the livestock industry. The etiological agent is a virus from the Flaviviridae family, genus Pestivirus, which is globally distributed with a prevalence of 60-85% in South American cattle herds. A relevant characteristic of this disesase is to produce immunotolerant animals (persistently infected - PI) that would not detected and eliminate a large amount of viruses in the herd, being the primary source of viral transmission. Early viral detection in the herd would allow a more effective control program. Here, we compared traditional molecular techniques (conventional PCR and Real-time PCR) to one of the latest generation (droplet digital PCR, ddPCR) which has shown significant advantages over the aforementioned ones. Serum samples from 46 animals, previously tested for BVD using conventional PCR and including both positive and negative results, were used for this comparison. Additionally, the NADL BVDV reference strain and a synthetic plasmid were used as positive controls, while non-template controls were used as negative controls. ddPCR showed higher sensitivity and precision for the detection and quantification of BVDV. ddPCR Limit of Detection was 0.24 copies/µL (286.3 × 10^-6 ηg/µL) for NADL BVDV reference strain, outperforming previous methods by an order of magnitude. The results showed that droplet digital PCR is a robust tool, with higher sensitivity, specificity and reproducibility, and it can be helpful when used in BVD control programs using pools of serum or bulk milk tanks.