Rapid and Sensitive Fluorescent RT-RAA Assay for the Detection of a Panel of Six Respiratory Viruses

Background: Rapid pathogen detection is crucial for the timely containment of outbreaks, particularly for respiratory infectious diseases which are highly transmissible and possess high epidemic potential. Methods: We developed a sensitive reverse transcription recombinase-aided amplification (RT-RAA) assay for the rapid detection of six common respiratory viruses: respiratory syncytial virus type A (RSV A), influenza A virus (Flu A), influenza B virus (Flu B), human parainfluenza virus (HPIV), SARS-CoV-2 and adenovirus (ADV). The assay employs a single, standardized protocol for the on-demand detection of any one of the six targets. Its performance was validated using nucleic acid standards and clinical pharyngeal swab specimens. Results: The assay enables rapid detection within 20 min at 39 °C using a portable, self-powered device. It demonstrated high sensitivity, with detection limits below 103 copies/mL for all targets and as low as 101 copies/mL for ADV. Cross-reactivity testing with 21 other pathogens confirmed excellent specificity. Validation with 85 clinical samples showed 100% concordance with RT-PCR, while offering significantly faster results and enhanced portability compared to RT-PCR. Conclusions: This sensitive, specific, and user-friendly RT-RAA assay provides a robust tool for rapid detection of respiratory viruses, particularly suitable for deployment in resource-limited settings and point-of-care testing during outbreaks.