Visual CRISPR-Cas12a Rapid Detection of Gyrovirus galga1

The global emergence of gyrovirus galga1 (GyVg1) across diverse regions and species un-derscores an urgent demand for rapid diagnostics. This study aimed to engineer a field-deployable diagnostic platform for rapid pathogen detection. We established two visual detection methods by integrating recombinase-aided amplification (RAA) and CRISPR/Cas12a technologies: RAA‒CRISPR/Cas12a combined with fluorescence and RAA‒CRISPR/Cas12a combined with lateral flow strips. By systematically optimizing the reaction conditions, designed primers and crRNA enabled target recognition within 1 hour, and demonstrated no cross-reactivity with other relevant avian pathogens. RAA‒CRISPR/Cas12a combined with fluorescence achieved a detection limit of 2 copies/µL (10 copies/µL visually under UV), and RAA-CRISPR/Cas12a-lateral flow strips demonstrated a detection limit of 5×10² copies/µL. Clinical validation using 192 samples revealed ~10% positivity rates across both novel methods and fluorescence quantitative PCR, with high concordance in positive identifications. The results suggest that the two RAA‒CRISPR/Cas12a visual detection methods established in this study are highly efficient, specific and sensitive, and can be used for the rapid field detection of GyVg1, providing a cost-effective and powerful diagnostic tool for grassroot workers.