A portable, one-tube CRISPR/Cas12a-based biosensor for sensitive and colorimetric detection of norovirus in foods without pre-amplification

Norovirus (NoV) is a leading cause of nonbacterial acute gastroenteritis, primarily transmitted through contaminated food and water. Rapid, sensitive, and on-site detection is crucial for controlling NoV outbreaks and ensuring food safety. In this study, we developed a portable, one-tube CRISPR/Cas12a-driven colorimetric biosensor for the rapid and sensitive detection of NoV RNA without the need for pre-amplification. The biosensor integrates magnetic nanoprobes with gold nanoparticle (AuNP) bioprobes, connected via single-stranded DNA (ssDNA) linkers. Upon the specific recognition of NoV cDNA, the Cas12a protein’s trans -cleavage activity is activated, cleaving the ssDNA linkers and releasing AuNP bioprobes from the magnetic nanoprobes. This release results in a visually detectable red color change and enhanced UV–visible absorbance in the solution following magnetic separation. These changes can be easily detected using portable devices such as microplate readers or smartphones. This method achieves a detection limit of 10 copies/µL within 60 min, with no cross-reactivity from other viruses or bacteria. Validation using oyster samples showed perfect concordance with qPCR results. Designed for resource-limited settings, our biosensor can be adapted for detecting various pathogens by altering the target nucleic acids and crRNA sequences. This advancement offers significant potential for enhancing rapid diagnostics and outbreak management.