G4-NASBA: An ultra-fast, highly sensitive and portable isothermal detection system for diagnosing influenza A virus.

Upper respiratory tract infection viruses (URTIs) continue to pose a serious threat to public health. There is a pressing need for rapid, reliable nucleic acid-based point-of-care testing (POCT) to detect these pathogens. Here, we developed an ultra-fast, highly sensitive, and portable viral detection system integrating nucleic acid sequence-based amplification (NASBA) with a G-quadruplex-Thioflavin T (G4-ThT) fluorescence sensor for viral RNA detection. We validated this system using influenza A virus (IAV), a major URTI pathogen. Results demonstrated that the system was capable of detecting IAV RNA at concentrations as low as 3 copies/μL without interference from other viral RNAs within 15 min. Furthermore, we compared the G4-NASBA system with RT-qPCR using 101 clinical IAV samples to assess clinical utility. Results showed no significant statistical difference in detection performance between the methods, with 94.0 % concordance relative to commercial kits. In conclusion, the G4-NASBA system is a robust and reliable technique well-suited for URTI RNA detection. This method also holds potential for integration into nucleic acid extraction systems and could incorporate other fluorescent aptamers to achieve a fully automated, multi-channel POCT platform.