Amplified-free and rapid nucleic acid detection of respiratory viruses based on streptavidin-functionalized quantum dot microspheres and magnetic beads.

Respiratory virus outbreaks-including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus (IAV), and influenza B virus (IBV)-posed severe threats to global health and economies, presenting critical challenges for international public health systems that contain highly contagious pathogens. The development of rapid, sensitive, and accurate diagnostic methods is therefore essential. Here, we developed a novel amplification-free detection platform that leverages DNA-RNA hybridization mechanism, utilizing streptavidin-functionalized magnetic beads (SA-MBs) bound to biotinylated capture probes (MBs-CP) for specific recognition and capture of target RNA and streptavidin-functionalized quantum dot microspheres (SA-QDMs) bound to biotinylated reporter probes (QDMs-RP) for fluorescence signal amplification. Through systematic optimization of the detection platform we proposed, theoretical limits of detection (LoDs) of target RNA synthesized by SARS-CoV-2 (N and Orf1ab), IAV (M) and IBV (NA) were 0.80 pM, 0.68 pM, 0.77 pM, and 0.80 pM, respectively. Moreover, our platform showed high concordance with RT-PCR results and exhibited robust performance in distinguishing negative and positive clinical samples. The platform demonstrated exceptional sensitivity (100%), specificity (80-100%), no cross-reactivity with 15 common pathogens, diagnostic accuracy (93.33%-100%), and long-term storage stability (CV < 12%) for four targets RNA. By employing QDMs for fluorescence signal amplification, this platform established a nucleic acid detection method featuring high sensitivity, user-friendly operation, time efficiency (≤53.5 min), and low cost ($5.34/test).