Evaluation of digital PCR for the detection of bovine viral diarrhea virus in persistently infected cattle

Bovine Viral Diarrhea Virus (BVDV) is a significant pathogen of cattle, causing substantial economic losses. Identifying persistently infected (PI) animals is crucial for BVD control and prevention, with pooled ear-notch tissue testing being a standard screening method. Digital PCR (dPCR) is an emerging diagnostic tool that provides absolute quantification of animal pathogens in low abundant targets. This study evaluates the effectiveness of dPCR for BVD surveillance using field submissions of pooled ear-notch samples, comparing it to real-time quantitative reverse transcription PCR (qRT-PCR). The study compared performance using 107 pools of ear notches (4853 sample animals), including 76 BVD-positive pools (3543 sample animals) and 31 BVD-negative pools (1310 sample animals). Pools included those that contained PI animals detected by IHC (n = 25) and those with only virus detection that were negative by IHC (n = 51). Performance metrics evaluated included analytical and diagnostic sensitivity and specificity, limit of detection (LOD) and test agreement with qRT-PCR. Results showed that dPCR exhibited enhanced sensitivity and lower LOD compared to qRT-PCR, detecting as low as 0.6 viral copies/μL. dPCR achieved 100 % sensitivity (76/76) and 96.77 % specificity (30/31) compared to qRT-PCR and no detection of common bovine pathogens. The comparison between qRT-PCR and dPCR using Cohen's kappa coefficient in pooled samples was 0.98, indicating almost perfect agreement. In pooled ear-notch samples with qRT-PCR quantification cycle (Cq) values ranging from 30 to 33.99, the viral load ranged from 4.75 to 194.78 viral copies/μL. The study suggests that dPCR is a sensitive and specific method for detecting BVD in pooled ear-notches.