Fe-Co bimetallic nanozyme-amplified lateral-flow immunoassay for the sensitive detection of porcine rotavirus.

Porcine rotavirus (PoRV) causes severe viral diarrhea in piglets. Owing to the lack of specific antiviral therapies, rapid on-site detection is urgently required for swine disease control. Herein, a lateral-flow immunoassay (LFIA) amplified by Fe-Co bimetallic nanozyme was established for PoRV detection using two screened high-affinity anti-PoRV monoclonal antibodies. The peroxidase-like catalytic activity of Fe-Co nanozyme was employed for signal amplification on the immunochromatographic strip. The limit of detection of this LFIA was 100 TCID50/mL, which was two-fold more sensitive than conventional commercial colloidal-gold-based test strips. No cross-reactivity was observed against other common porcine pathogens, including porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), porcine sapelovirus (PSV), transmissible gastroenteritis virus (TGEV), porcine parvovirus (PPV), porcine reproductive and respiratory syndrome virus (PRRSV), and porcine pseudorabies virus (PRV). The intra-batch and inter-batch coefficients of variation were both below 10%, demonstrating favorable repeatability. The strip remained stable for at least 9 months under 4°C and ambient-temperature storage. When evaluated with 72 clinical pig fecal samples using RT-qPCR as the reference, the LFIA showed a sensitivity of 95.45%, specificity of 100%, and a total agreement rate of 98.61%, with a kappa value of 0.97 (95% CI: 0.891-1.000), indicating excellent agreement between the two methods. This Fe-Co nanozyme-amplified LFIA possesses high sensitivity, outstanding specificity, reliable stability and easy-to-operate characteristics. It can be applied for on-site PoRV screening in pig farms and facilitates early epidemic intervention of PoRV-associated diarrhea.