Visual Detection of Foodborne Viruses in Leafy Greens Using an AsCas12f-Split-G-Quadruplex Sensor

Abstract Foodborne viruses are responsible for a substantial burden of disease worldwide, with fresh produce serving as the most abundant vehicle for transmission. However, rapid detection in solid produce, such as leafy greens, remains underdeveloped, as conventional methods often require labor-intensive sample preparation and specialized instrumentation. Herein, we present a colorimetric CRISPR-split-G4 biosensor for rapid viral surveillance in leafy greens that combines reverse transcription recombinase polymerase amplification (RT-RPA), AsCas12f-mediated trans-cleavage, and a modular split-G-quadruplex (split-G4) reporter system. In this system, target recognition activates the trans-cleavage activity of AsCas12f, resulting in degradation of a blocker strand and subsequent assembly of a catalytically active G4-hemin DNAzyme that generates a visible signal easily detectable by the naked eye. Using norovirus as a model pathogen, the platform exhibited high analytical sensitivity and specificity, enabling reliable detection of viral particles with minimal instrumentation. To evaluate practical applicability, the assay was coupled with a simplified leaf-disk extraction workflow and successfully applied to contaminated romaine lettuce and baby spinach samples without requiring viral nucleic acid purification. The platform achieved detection limits of 1000 and 5000 viral copies per 100 mg of romaine lettuce and spinach tissue, respectively. From sample processing to visual readout, the entire assay was completed in approximately 2 h. By integrating isothermal amplification, CRISPR-based target recognition, and instrument-free signal generation into a streamlined workflow, this strategy provides a promising platform for decentralized food safety monitoring and establishes a flexible framework for the on-site detection of viral pathogens in complex food matrices.