PoRVのVp4遺伝子に対するドロップレットデジタルPCR検出法の確立

Diarrhea outbreaks in pigs occur most frequently during winter, porcine rotavirus (PoRV) is one of the important diarrheal diseases. Droplet digital polymerase chain reaction (DDPCR), a detection method that can perform absolute quantification of genes. The study aimed to diagnose PoRV infection using a probe-based DDPCR. 10 2-day-old piglets with mild diarrhea were obtained from a commercial pig farm. No PoRV was detected in the anal swab using colloidal gold test strips. To verify the results of the colloidal gold test strips, RT-qPCR was performed, which identified PoRV in six piglets. Given the limited sensitivity of colloidal gold test strips and RT-qPCR, we developed a DDPCR assay targeting the PoRV Vp4 gene for enhanced detection. The DDPCR assay demonstrated optimal performance at a primer:probe concentration of 400:400 nM and an annealing temperature of 57 °C. It achieved a minimum detection limit of 0.21 copies/μL, the detection sensitivity has been enhanced by 100 times compared to RT-qPCR. Using the established DDPCR detection method, the four samples that tested negative by RT-qPCR were re-tested, and all were found to be PoRV-positive, indicating that the sensitivity of DDPCR was higher than that of RT-qPCR. This study highlights its potential as a valuable tool for early clinical diagnosis and disease control in piglets.