PfAgo-mediated a rapid and visual detection assay for porcine circovirus type 3 based on recombinase-aided...

Porcine circovirus type 3 (PCV3) is widely distributed in swine populations and has been associated with diverse clinical manifestations. Its broad tissue tropism and variable clinical presentation pose challenges for rapid and reliable diagnosis, particularly under field or resource-limited conditions.

Here, we report a rapid nucleic acid detection assay that integrates recombinase-aided amplification (RAA) with Pyrococcus furiosus Argonaute (PfAgo) for PCV3 detection. The sealed-tube, two-stage RAA-PfAgo assay targets a highly conserved 41-bp region within the ORF2 gene and completes amplification and detection within 35 min. The assay achieved a limit of detection of 1 copy/μL with high specificity and no cross-reactivity toward other swine pathogens. To enhance result interpretability, a semi-quantitative cutoff-based classification strategy inspired by ELISA analysis was introduced, enabling robust discrimination between positive and negative samples under variable background conditions. Evaluation using 107 clinical samples showed complete concordance with a reference TaqMan qPCR assay.

By combining isothermal amplification with programmable nuclease-mediated signal generation and an interpretability-oriented analytical framework, the proposed RAA-PfAgo assay provides a rapid, sensitive, and instrument-flexible platform for PCV3 detection. This study highlights the potential of PfAgo-based diagnostics for practical clinical and on-site applications beyond sensitivity-driven proof-of-concept demonstrations.