Development and Validation of a Multiplex TaqMan Real-Time PCR Assay for Simultaneous Detection of PEDV Genotypes G1, S-INDEL, and G2

Porcine epidemic diarrhea virus (PEDV) is a major pathogen responsible for severe diarrhea, dehydration, and high mortality in neonatal piglets, continually threatening global swine production. Rapid differentiation of its major genotypes (classical G1, variant G2, and recombinant S-INDEL) is vital for molecular epidemiology and effective disease control, yet existing approaches rely mainly on time-consuming sequencing and phylogenetic analysis of the S gene. To overcome this limitation, we developed a novel triplex TaqMan-based real-time PCR assay for rapid detection and differentiation of the three PEDV genotypes. The assay demonstrated high sensitivity, with the lowest detection limit of 10² copies/μL, and strong specificity, showing no cross-reactivity with six other common swine pathogens (TGEV, PDCoV, PoRV, PRRSV, CSFV, and PRV). It also exhibited excellent reproducibility, with both intra- and inter-assay coefficients of variation maintained below 1.5%. In clinical validation, the assay showed 100% concordance with results obtained from S gene sequencing and phylogenetic analysis. Furthermore, testing of 160 clinical samples revealed cases of co-infection involving G2 and S-INDEL strains. In conclusion, this rapid, specific, and reproducible assay provides a reliable tool for routine molecular diagnosis, facilitating large-scale epidemiological surveillance and enabling genotype-informed control strategies against PEDV.