Gyrovirus galga1 (GyG1) and Gyrovirus homsa1 (GyH1) are the second and third most common gyroviruses identified, respectively, after chicken anaemia virus. They were first reported in 2011 and are currently prevalent worldwide. However, limited research on these pathogens and a lack of prevention and control strategies have necessitated the establishment of a rapid diagnostic technique to address new challenges in infectious diseases. Recombinase acid amplification (RAA) combined with CRISPR - Cas12a or CRISPR - Cas13a technology has major advantages for highly sensitive and rapid diagnosis. Specific targets can activate CRISPR-Cas trans-cleavage activity, resulting in non-specific cleavage of single-stranded DNA by the CRISPR - Cas12a complex and RNA cleavage by the CRISPR - Cas13a complex. In this study, for the first time, we combined RAA-based CRISPR - Cas12a and CRISPR - Cas13a systems for simultaneous differential diagnosis of GyG1 and GyH1 infection. The results showed that dual fluorescence channel RAA-based CRISPR - Cas12a/Cas13a technology could detect GyG1 and GyH1 within one hour, with a minimum detection limit of 1.5 copies of the target DNA standard/µL and no cross-reactivity with other avian pathogens. In addition, this method could be used for clinical detection, with the results exhibiting high consistency with those obtained by qPCR. These findings demonstrate that our RAA-based CRISPR - Cas12a/Cas13a dual-channel detection system can detect two different subtypes of gyrovirus in a sample with good specificity and high sensitivity, improving the detection efficiency and providing a new technique for the study of viral infection dynamics.

Gyrovirus galga1 (GyG1), an emerging pathogen within the Anelloviridae family, exhibits expanding host diversity and zoonotic potential, yet its epidemiological patterns and genomic determinants remain poorly characterized. To address this critical knowledge gap, we conducted the first comprehensive molecular epidemiological investigation of GyG1 in Guangxi, China (2020-2025). The result revealed three key advances: (1) Molecular-epidemiological evidence of winter-spring seasonality and age-dependent transmission, with breeder chickens showing biphasic peaks (1-6 and > 21 weeks) contrasting broilers' progressive carriage; (2) Genomic characterization revealing VP1-VP3 as nuclear-localized hydrophilic proteins, identifying VP1 nuclear targeting as a therapeutic vulnerability; (3) Functional annotation of recombination-driven evolution, including a temporal paradox in strain KX708518's ancestry. This study elucidates GyG1's ecological dynamics in a high-density poultry region and provides critical data for One Health surveillance and vaccine development, addressing a key gap in gyrovirus countermeasures.

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.