Development of a Duplex-ddPCR assay for accurate quantification of pseudorabies virus through systematic optimization of amplification bias.

Pseudorabies (PR), caused by the pseudorabies virus (PRV), is highly contagious. Although qPCR is widely used for viral DNA detection, it struggles with low-level DNA identification and precise quantification. To address these issues, droplet digital PCR (ddPCR) has emerged as a more advanced method for detecting pathogens and providing absolute quantification of nucleic acids. The study introduces a ddPCR assay for accurate PRV quantification, addressing the challenges posed by the high GC content of the PRV genome. By optimizing factors such as primer and probe concentrations, annealing conditions, denaturation time, and cycle number, the assay overcomes limitations of traditional PCR techniques. The optimized ddPCR assay showed a wide linear dynamic range, with well-defined limits of blank (LOB) and detection (LOD). Testing confirmed the method's reproducibility, demonstrating its stability and reliability. This study provides key insights into optimizing ddPCR for GC-rich templates and serves as a useful reference for future experiments.