Development of a High-Resolution Melting (HRM)-Based Multiplex Real-Time PCR Assay of PEDV, TGEV, PoRV, PDCoV, PRV, and PRRSV.

Six key viral pathogens-porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine rotavirus (PoRV), porcine deltacoronavirus (PDCoV), pseudorabies virus (PRV), and porcine reproductive and respiratory syndrome virus (PRRSV)-inflict substantial economic losses on the global swine industry. Their frequent cocirculation and the resulting mixed infections severely complicate rapid and accurate diagnosis. To overcome this challenge, we developed a multiplex real-time PCR assay integrated with high-resolution melting (HRM) analysis for their simultaneous detection and differentiation. Specific primer sets were designed and optimized to target conserved regions of the PEDV-M, TGEV-N, PoRV-VP6, PDCoV-M, PRV-gE, and PRRSV-ORF5 genes. Reaction conditions and HRM parameters were refined, ensuring specific amplification and distinct melting profiles for all six viruses. The assay exhibited excellent specificity, showing no cross-reactivity with other common porcine pathogens. Limits of detection (LODs) were established at 10 copies/μL for five viruses (PEDV, TGEV, PoRV, PDCoV, and PRRSV) and 100 copies/μL for PRV. Amplification efficiency ranged from 90% to 102% (R2 ≥ 0.98). High reproducibility was confirmed by intra- and interassay coefficients of variation (CVs) of <3% for cycle threshold (Ct) values and <0.5% for melting temperature (Tm) values. Clinical validation of 248 field samples demonstrated that detection rates for the six pathogens were 16.0%-27.3% higher (an average of 18.1%) than those of conventional PCR assays. Collectively, these findings confirm that the HRM-based multiplex real-time PCR assay is a rapid, sensitive, specific, and highly reproducible tool for detecting and differentiating major porcine viral pathogens. This methodology offers a valuable molecular diagnostic platform, supporting early diagnosis, epidemiological surveillance, and enhanced control of swine viral diseases.