Double-color ELISA based on fluorescence hybrid chain reaction for simultaneous detection of PEDV and PoRV-A

Porcine epidemic diarrhea virus (PEDV) and group A porcine rotavirus (PoRV-A) are two predominant pathogens leading to viral diarrhea in piglets. These two viruses often result in clinical mixed infections and exhibit indistinguishable clinical symptoms and pathological lesions, making them difficult to differentiate in routine diagnosis. Conventional ELISA suffers from low detection sensitivity and single-pathogen detection, while multiplex nucleic acid diagnostic methods rely on cumbersome nucleic acid extraction, restricting their on-site application in grassroots pig farms. Herein, an extraction-free double-color fluorescent HCR-ELISA platform is constructed for the simultaneous and ultrasensitive detection of PEDV and PoRV-A. A novel freeze-thaw combined electrostatic adsorption strategy was developed to prepare bifunctional AuNPs-Initiator-mAb probes, which rapidly integrate viral antigen recognition and HCR cascade signal initiation on a single nanoparticle. Two sets of spectrally isolated FAM/Cy3-labeled HCR hairpin probes were designed to realize crosstalk-free dual fluorescent signal amplification. The established method displayed no cross-reactivity with six common swine pathogenic viruses, with limits of detection (LOD) of 1.3 × 10¹ TCID₅₀/mL for most PEDV strains and 3.2 × 10¹ TCID₅₀/mL for predominant PoRV-A genotypes, and intra- and inter-batch coefficients of variation (CV) were all less than 5%, reflecting favorable specificity, sensitivity and reproducibility. Clinical detection of 191 pig fecal samples achieved total diagnostic coincidence rates of 96.3% (PEDV) and 97.9% (PoRV-A) compared with commercial RT-qPCR. Integrating enzyme-free isothermal HCR amplification with high-throughput ELISA, the double-color HCR-ELISA overcomes the limitations of traditional detection methods and possesses great promise for high-throughput screening of swine viral diarrhea in grassroots pig farms.