Argonaute-based dual lateral flow test for SARS-CoV-2 and influenza a virus

• A novel dual nucleic acid lateral flow test based on Argonaute (Ago-LFT) has been developed. • The Ago-LFT utilized a variety of secondary antibodies from different animal sources to specifically capture Ago-cleaved reporters. • The Ago-LFT provided a naked-eye readout and user-friendly operation, making it suitable for point-of-care testing (POCT) applications. • In clinical validation, the Ago-LFT demonstrated superior sensitivity and specificity compared to both Ago-based fluorescent detection and commercial RT-qPCR. The lateral flow test (LFT) is crucial for community screening and early warning of infectious pathogens due to its portability and simplicity. However, antigen-based LFTs have relatively low sensitivity, while nucleic acid-based LFTs are limited in specificity and multiplexing capabilities, thus requiring improvements in accuracy and efficiency. In this study, we developed a dual lateral flow assay utilizing a thermophilic Argonaute, enabling the detection of multiple nucleic acids on a single strip with a sensitivity of 3 copies/μL and high specificity. Clinical validation showed 100 % concordance with commercial RT-qPCR results. This method not only preserves the high sensitivity of nucleic acid detection but also provides the benefits of portability, dual-target detection, and visual readout, highlighting its potential for point-of-care applications.