PfAgo-mediated a rapid and visual detection assay for PCV3 based on recombinase-aided amplification

Porcine circovirus type 3 (PCV3) is associated with a myriad of clinical presentations, from Porcine Dermatitis Nephropathy Syndrome (PDNS) to porcine respiratory disease complex (PRDC), and it has been reported in domestic swine populations globally at low to moderate prevalence. Rapid, sensitive, accurate and specific detection is important for early accurate diagnosis of PCV3. Here, we introduce a novel assay method developed by integrating Recombinase-aided amplification (RAA) with Pyrococcus furiosus Argonaute ( Pf Ago), specifically targeting a 41-bp highly conserved region of the ORF2 gene of PCV3. The RAA- Pf Ago system including amplification and detection procedures can be completed within 45 min with the limit of detection (LOD) of 1 copy/µL, and the results were superficially interpreted by the intensity of the collected fluorescence signal. The RAA- Pf Ago system can accurately identify PCV3 infection without cross-reacting with other swine clinical pathogens. Validation of this method was performed on 30 clinical isolates, demonstrating a 95.0% accuracy rate when compared to the PCV3 Taqman qPCR assay. Our findings highlight the potential of RAA- Pf Ago method as an exceptional diagnostic tool characterized by high speed, simplicity, accuracy, and sensitivity.