Development of a Rapid Recombinase Polymerase Amplification Assay for Avian Polyomaviruses

Budgerigar fledgling disease, caused by avian polyomavirus (APV), is an immunosuppressive condition in budgerigars that typically manifests as abdominal swelling, absence of down feathers on the dorsal and ventral regions, subdermal bleeding in nestlings, and high mortality. A new APV detection relied on real-time quantitative recombinase polymerase amplification (real-time RPA) targeting the large T antigen gene using specific probe and primers. Detection can be accomplished within 25 minutes at 41°C, with this detection threshold of 2.56 × 10 2 copies/μL. The real-time RPA assay specific detected APV and did not show cross-reactivity with avian infectious bronchitis virus (IBV), psittacine beak and feather disease virus (PBFDV), newcastle disease virus (NDV) and H9N2 avian influenza virus(H9N2). Intra-assay repeatability testing showed that the coefficients of variations (CVs) were all below 16%, while inter-assay repeatability testing showed that the CVs were all below 12%. In 63 parrot cases suspected of APV-associated mortality, tissue specimens were examined using real-time RPA alongside qPCR assays. Both detection methods showed identical positive rates, indicating complete agreement in positive/negative classification (κ coefficient = 1). Taken together, a real-time RPA method for APV screening was established in this study.