Development of a droplet digital PCR for detection and quantification of porcine epidemic diarrhea virus.

Porcine epidemic diarrhea, a disease caused by porcine epidemic diarrhea virus (PEDV), results in large economic losses to the global swine industry. To manage this disease effectively, it is essential to detect PEDV early and accurately. We developed a sensitive and accurate droplet digital PCR (ddPCR) assay to detect PEDV. The optimal primer-to-probe concentration and melting temperature were identified as 300:200 nM and 59.2°C, respectively. The specificity of the ddPCR assay was confirmed by negative test results for common swine pathogens. The detection limit for the ddPCR was 0.26 copies/μL, which is a 5.7-fold increase in sensitivity compared to that of real-time PCR (rtPCR). Both ddPCR and rtPCR assays exhibited good linearity, although ddPCR provided higher sensitivity for clinical detection compared to that of rtPCR. Our ddPCR methodology provides a promising tool for evaluating the PEDV viral load when used for clinical testing, particularly for detecting samples with low-copy viral loads.