A lyophilized paper-based platform integrating RT-RPA and CRISPR/Cas12a for smartphone-assisted point-of-care detection of norovirus in environmental and food samples

Norovirus (NoV) represents a leading cause of foodborne illness worldwide and is the predominant agent of non-bacterial acute gastroenteritis. The development of rapid, highly sensitive, specific, and point-of-care detection methods is critical for the protection of public health and food safety. In this study, we developed a lyophilized paper-based microfluidic analytical device (μPAD) that integrates reverse transcription recombinase polymerase amplification (RT-RPA) with CRISPR/Cas12a technology. By incorporating a smartphone-assisted portable fluorescence detection device, the platform achieves a high level of integration, simplicity, and intelligence. It demonstrates a detection limit for NoV as low as 1 copy/μL within 45 min, with no cross-reactivity against other common microorganisms or the human genome. The fluorescence intensity on the chips exhibits a strong linear relationship with NoV RNA concentration, enabling accurate quantification. Moreover, the lyophilized RPA-CRISPR paper chips retain stability for up to 8 weeks at room temperature, underscoring their suitability for point-of-care applications. When applied to real food and environmental samples for NoV detection, the platform showed 100% concordance with RT-qPCR and maintained a detection sensitivity of 1 copy/μL. This work provides a robust approach for rapid, sensitive, and on-site detection of norovirus, and the platform architecture holds promise for broader application in detecting other microorganisms.