G-Quadruplex DNAzyme-Assisted CRISPR/Cas12b Assay for Label-Free Detection of Foodborne Pathogens by the Naked Eye

Abstract

Rapid, accurate, and on-site detection of foodborne pathogens is of great significance to ensure food safety. The strategies of clustered regularly interspaced short palindromic repeats (CRISPR) combined with test strips or gold nanoparticles offer promising options for on-site detection. However, these methods have different defects such as complex labeling, high cost, and susceptibility to aerosol contamination. Herein, we report a G-quadruplex DNAzyme-assisted CRISPR/Cas12b-dUTP-LAMP visual method for rapid and sensitive detection of foodborne pathogens. In this method, it was found that self-assembled DNA nanostructures, G-quadruplexes, can be cleaved by the trans-cleavage activity of AapCas12b. In order to increase the sensitivity and avoid aerosol contamination, dUTP-loop-mediated isothermal amplification (LAMP) and mineral oil were introduced. In this way, the results can be directly distinguished with the naked eye through the 3,3′,5,5′-tetramethylbenzidine (TMB)/H₂O₂ color reaction. The proposed method can detect 29 copies/μL of Salmonella genomic DNA with high specificity. Overall, this simple visual method may serve as a potential tool for on-site detection applications.