Rapid, sensitive, and highly specific diagnosis of respiratory syncytial virus using recombinase polymerase amplification-based biosensor and fluorescence detection

Respiratory syncytial virus (RSV) is a major cause of acute lower respiratory tract infections in children, highlighting the need for rapid and accessible molecular detection. Here, we developed two recombinase polymerase amplification (RPA)-based assays for the detection and differentiation of RSV-A and RSV-B, combining fluorescence readout or lateral flow biosensor (LFB) visualization, termed RSV-RPA-FLU and RSV-RPA-LFB, respectively. The workflow can be completed within 40 min, including sample preparation for 15 min, isothermal amplification for 20 min, and result interpretation within 5 min. Analytical sensitivity testing showed that the RSV-RPA-FLU assay detected both RSV-A and RSV-B at 10⁴ copies/μL. The RSV-RPA-LFB assay showed higher sensitivity, with detection limits of 6.05 × 10³ copies/μL for RSV-A and 7.80 × 10² copies/μL for RSV-B. Specificity analysis against 11 non-RSV respiratory pathogens showed no cross-reactivity. Preliminary evaluation using simulated clinical samples further supported the feasibility of both assays in complex sample matrices. These results indicate that the RSV-RPA-FLU and RSV-RPA-LFB assays provide rapid, specific, and operationally simple analytical platforms for RSV-A/RSV-B detection and differentiation, holding great potential for point-of-care RSV diagnosis and global prevention and control.