A triplex-readout CRISPR-Cas12a multimodal biosensing platform for point-of-care detection of avian influenza H5N1

The highly pathogenic avian influenza A (H5N1) virus poses a significant zoonotic threat to public and animal health. Conventional detection methods often face limitations in complexity, time, and equipment requirements. In this study, we report a rapid and lightweight-equipment triplex diagnostic platform for H5N1 detection, integrating locked nucleic acid (LNA)-assisted target recognition, toehold-mediated strand displacement for signal amplification, and the high specificity of the CRISPR-Cas12a system. The developed assay achieved a detection limit of 3.7 × 10² copies/μL for H5N1 pseudovirus RNA within a 60-minute core detection process, and exhibited excellent specificity without cross-reactivity to other influenza subtypes or coronaviruses. Moreover, it successfully identified H5N1 in clinical swab samples, yielding consistent results across three independent readout formats: fluorescence, lateral flow strip, and a portable glucose meter. With advantages of low cost, rapid operation, and multimodal verification capability, this diagnostic system is well suited for point-of-care screening in resource-limited settings and represents a promising tool for frontline outbreak response.