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.