Development of a rapid, sensitive and visual reverse transcription recombinase-aided amplification coupled with lateral flow dipstick assay for influenza B virus detection.

INTRODUCTION: Influenza B virus (IBV) is a major etiological agent of seasonal influenza, imposing a significant global health burden. Rapid and accurate detection is crucial for epidemic control, but conventional methods are limited by complex equipment or long turnaround times. METHODS: Herein, we developed a reverse transcription recombinase-aided amplification assay combined with lateral flow dipstick (RT-RAA-LFD) for specific IBV detection, targeting the conserved ns gene. RESULTS: Following primer screening, the optimal primer pair F2/R4 was identified. The assay was optimized to react at 39°C for 20 min with a detection volume of 15 μL. Analytical validation demonstrated high sensitivity (50 copies/mL) and no nonspecific cross-reactivity with other common respiratory viruses, including influenza A virus, SARS-CoV-2, parainfluenza virus and adenovirus. Clinical performance evaluation revealed 100% concordance with the gold standard PCR assay. DISCUSSION: Collectively, the established RT-RAA-LFD assay is rapid, specific, sensitive and easy to operate without relying on sophisticated instruments, making it a promising tool for point-of-care testing (POCT) and field surveillance of IBV infections.