Development of a Visual Rapid Assay for Novel Goose Astrovirus Detection Based on RT-MIRA -PfAgo

Goose astrovirus genotype 2 (GAstV-2) is an important pathogen associated with gosling gout, and rapid detection is useful for early diagnosis and field surveillance. In this study, a visual assay for GAstV-2 detection was developed by combining one-step reverse transcription multienzyme isothermal rapid amplification (MIRA) with the nucleic acid cleavage activity of Pyrococcus furiosus Argonaute (PfAgo). MIRA primers and specific guide DNAs were designed based on a conserved region of the GAstV-2 ORF1b gene, and the PfAgo reaction conditions were optimized. The optimal reaction contained 1.0 μM gDNA, 0.6 μM PfAgo, and 1.0 mM MnCl₂. Using recombinant pUC57-ORF1b plasmid DNA as the template, the lowest detectable plasmid concentration under the tested conditions was 1.0 × 10⁰ copies/μL. In the specificity assay, only GAstV-2 produced a positive signal, with no cross-reaction observed with GAstV-1, Tembusu virus, H9-subtype avian influenza virus, goose circovirus, fowl adenovirus serotype 4, or goose parvovirus. The assay was further tested with 23 clinical samples suspected of GAstV-2 infection. In a preliminary evaluation of 23 clinical samples, the RT-MIRA-PfAgo results were concordant with those obtained by conventional RT-PCR and RT-qPCR. Overall, the RT-MIRA-PfAgo assay provided sensitive and specific GAstV detection within a short time, without requiring programmed thermal cycling or an expensive real-time PCR instrument for routine endpoint detection. This method may be useful for GAstV-2 detection in basic laboratories and field settings.