In recent years, pigeon rotavirus A (PiRVA) infection, an important emerging disease, has posed a major threat to the healthy development of the pigeon industry and public health. Therefore, developing an accurate, rapid and convenient detection method for this virus is vital for monitoring and early diagnosis of the disease. In this study, on the basis of the ORF sequence characteristics of the PiRVA VP6 gene, crRNA and reverse transcription recombinase-aided amplification (RT-RAA) primers were designed. On the basis of the CRISPR/Cas12a system, for the first time, the RT-RAA-CRISPR/Cas12a rapid detection method of PiRVA was established by combining RT-RAA and lateral flow strips. This method could specifically detect PiRVA, and there was no cross-reaction with other common viruses originating from pigeons. The minimum detection limit was 16.8 copies/μL, and the results of the intrabatch and interbatch repeated tests were consistent. Moreover, the method established in this study and the previously established common PCR method were used to analyse 56 clinical tissue samples from racing pigeons and domestic pigeons collected in 2025. The positive rates of racing pigeon and domestic pigeon samples detected by PCR were 17.6% and 12.8%, respectively, and the positive rates of racing pigeon and meat pigeon samples detected by the RT-RAA-CRISPR/Cas12a method were 23.5% and 17.9%, respectively, indicating that PiRVA infection occurs in both racing pigeon and domestic pigeon populations in China. In summary, the PiRVA RT-RAA-CRISPR/Cas12a detection method established in this study has good specificity, sensitivity, and reproducibility, and allows visualization of the results, which can be used for field applications. This study provides technical support for epidemiological surveillance and etiological research on PiRVA.

Avian metapneumovirus subtype C (aMPV/C) has re-emerged as a widespread pathogen in China, causing hydrosalpinx fluid syndrome (HFS) in laying ducks and resulting in substantial economic losses, highlighting the urgent need for a reliable immunological detection method. In the present study, a non-invasive indirect enzyme-linked immunosorbent assay (iELISA) was developed and optimized for detecting aMPV/C-specific immunoglobulin Y (IgY) in egg yolks of laying ducks, using purified novel aMPV/C as the coating antigen. Checkerboard titration established the optimal conditions as an antigen concentration of 1.25 μg/mL and an IgY dilution of 1:200. The cut-off value, determined by receiver operating characteristic curve analysis, was 0.3268, enabling a detection limit of 1:3200 for IgY antibodies. The assay showed no cross-reactivity with IgY positive for other duck pathogens, and both intra-assay and inter-assay coefficients of variation were less than 10%, demonstrating good specificity and reproducibility. Clinical evaluation of 151 duck egg yolk samples yielded an overall aMPV/C-specific IgY positivity rate of 58.3% (88/151). The concordance rate between the iELISA and an indirect immunofluorescence assay (IFA) was 97.8% (45/46). These results indicate that the established iELISA for detecting aMPV/C-specific IgY is a reliable and practical tool for large-scale detection of aMPV/C exposure in clinical duck egg yolk samples.