Isothermal cross-priming amplification for field-deployable norovirus surveillance: a cost-effective and rapid...

Norovirus remains a leading cause of foodborne gastroenteritis globally. While isothermal amplification technologies offer advantages over traditional reverse transcription quantitative PCR (RT-qPCR) in terms of speed and portability, there is a lack of domestically developed isothermal amplification systems for norovirus in China. Using a domestic patented cross-priming amplification (CPA) technology, we established CPA systems targeting the predominant norovirus genotypes GII.3, GII.4, and GII.17. The systems were evaluated using 224 fecal samples with confirmed genotypes via sequencing. Our lab-developed CPA (Lab-CPA) systems demonstrated high sensitivity, detecting 98.0% (49/50) of GII.3, 100% (80/80) of GII.4, and 96.9% (31/32) of GII.17 samples, with 100% specificity across all genotypes. Compared to certified clinical RT-qPCR kits, the total percent agreement was 98.7%, 99.3%, and 96.9% for GII.3, GII.4, and GII.17, respectively, accompanied by a significant linear correlation in threshold times. Crucially, the Lab-CPA systems reduced processing time by 44-60 min compared to RT-qPCR. Successful integration into the commercial EasyNAT platform using all-in-one cartridges further streamlined the workflow while maintaining accuracy. Our CPA systems offer a rapid, cost-effective, and streamlined diagnostic solution, ideally suited for point-of-care testing in resource-limited primary healthcare environments.IMPORTANCENorovirus remains a critical public health threat, driving demand for rapid, deployable diagnostics. While isothermal amplification offers theoretical advantages over reverse transcription quantitative PCR, the absence of domestically developed systems in China has created a reliance on imported assays, limiting point-of-care testing capabilities. This study addresses that gap by establishing a novel cross-priming amplification system tailored to China's predominant norovirus genotypes. Achieving high sensitivity/specificity and significantly reducing processing time, this work validates a robust, field-adaptable alternative to conventional methods. Furthermore, successful integration with the commercial EasyNAT platform demonstrates immediate translational potential. By providing a cost-effective, high-performance domestic solution, these findings significantly enhance decentralized surveillance and emergency response capacity for norovirus infections in resource-limited settings.