The global emergence of gyrovirus galga1 (GyG1) across diverse regions and species underscores the urgent need for rapid diagnostic methods. 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 technology: ‌RAA‒CRISPR/Cas12a combined with fluorescence and RAA‒CRISPR/Cas12a combined with lateral flow strips. By systematically optimizing the reaction conditions, the designed primers and crRNA enabled target recognition within 1 hour, and the system exhibited 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 combined with lateral flow strips exhibited 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 identification. The results suggest that the two RAA‒CRISPR/Cas12a-based visual detection methods established in this study are highly efficient, specific and sensitive and can be used for rapid field detection of GyG1, providing a cost-effective and powerful diagnostic tool for field workers.

To Cite This Article: Yu D, Xie Z, Zhang Y, Xie Z, Luo S, Xie L, Li M, Fan Q, Zeng T, Zhang M, Li X, Wei Y, Wu A and Wan L 2026. Visual CRISPR/Cas12a-mediated rapid detection of gyrovirus galga1. Pak Vet J, 46(2): 438-445. http://dx.doi.org/10.29261/pakvetj/2026.032