Colorimetric Detection of Antimicrobial Resistance from Food Processing Facilities Using a CRISPR System

Abstract

The overuse of antibiotics has led to considerable increases of antibiotic resistance genes (ARGs) that have spread throughout the entire food supply chain. ARGs often transfer from processing facilities to food, causing major public health concerns. The current standard for detecting ARGs typically depends on quantitative polymerase chain reaction (qPCR) and gene sequencing. However, the reliance on experienced personnel and complicated readout equipment substantially inhibits the expansion of ARGs testing in nonlaboratory settings. Improved on-site testing will help monitor the spread of ARGs contamination to ensure food safety and address public health concerns. Herein, we developed a CRISPR-Cas12a-based assay for the colorimetric detection of ARGs in washing water collected from a food processing plant. In our assay, DNA-functionalized gold nanoparticles (AuNPs) were cross-linked with a ssDNA cross-linker. Target-induced Cas12a trans-cleavage was utilized for degradation of the cross-linkers, shifting the optical properties of the AuNPs to produce a facile visual readout. Without DNA amplification, we were able to detect three representative ARGs with a detection limit of 5 nM or lower. In addition, our assay was extended to a more complex medium, where as few as 103 gene copies in washing water from a fruit washing facility were visually detected. Based on our results, our method is both highly specific and sensitive. Due to the affordability and simplicity of our assay, this method can improve ARGs detection to monitor and prevent the immense spread of antimicrobial resistance among the food supply chain.