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Development of a High-Resolution Melting (HRM)-Based Multiplex Real-Time PCR Assay of PEDV, TGEV, PoRV, PDCoV, PRV, and PRRSV
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DOI：10.1155/tbed/1574555.png)
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Abstract
En 中文
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.
Keywords:
high-resolution melting (HRM)
major porcine viral pathogens
multiplex real-time PCR
simultaneous and differential detection
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