A red/blue bicolor lateral flow immunoassay for simultaneous detection of two enteroviruses based on duplex RT-LAMP

Norovirus and human adenovirus are the main pathogens causing acute gastroenteritis (AGE) worldwide. Their highly contagious nature, significant disease burden, similar symptoms, and frequent co-infection necessitate a differential diagnostic tool to guide epidemic control and support antimicrobial stewardship by rapidly ruling out bacterial etiologies.

This study developed an integrated dual reverse transcription loop-mediated isothermal amplification (RT-LAMP) and dual-color lateral flow immunoassay (LFIA) technology for rapid, sensitive, and visual synchronous detection of norovirus genogroup II (NoV GII) and human adenovirus F41 (HAdV F41) in human feces. LAMP primers for the two viruses were labeled with FAM/biotin and TAMRA/biotin, respectively. Combined with a dual-color gold nanomaterial signaling system (red AuNPs for test lines, blue AuNFs for control line), the assay allows for intuitive visual interpretation.

The method demonstrated good sensitivity and specificity, with detection limits of 5.8 copies/μL for NoV GII and 63 copies/μL for HAdV F41, and no cross-reactivity with common non-target pathogens. The entire reaction process requires only a constant temperature of 65°C and can be completed within 45 min.

The clear dual-color interpretation system, user-friendly operation, and excellent performance make it a highly promising tool for first-line screening and point-of-care diagnosis of AGE.