Abstract

Porcine rotavirus (PoRV), porcine epidemic diarrhea virus (PEDV), and porcine deltacoronavirus (PDCoV) are major pathogens responsible for porcine diarrheal diseases, and their accurate identification is critical for disease control in swine production. However, overlapping clinical symptoms and pathological manifestations pose significant challenges for differential diagnosis. We developed a highly sensitive and specific triplex RT-qPCR assay to simultaneously detect and distinguish these three pathogens. Specific primers and probes were designed and screened based on conserved regions of the NSP5 gene of PoRV, the S gene of PEDV, and the S gene of PDCoV. The reaction system was optimized for primer concentrations, probe concentrations and annealing temperature. The assay demonstrated excellent specificity with no cross-reactivity to other common swine pathogens, a detection sensitivity of 10 copies/µL, and intra-batch and inter-batch coefficients of variation (CV) below 1%. Additionally, the method exhibited strong tolerance to inhibitors in complex sample. Parallel testing of 124 clinical samples using this triplex assay and singleplex RT-qPCR commercial kits showed complete concordance in detection rates for PoRV, PEDV, and PDCoV. Our study presents a robust triplex RT-qPCR method that provides a rapid, sensitive, and reliable tool for the differential diagnosis of porcine diarrheal pathogens.