G-Quadruplex-Probing CRISPR-Cas12 Assay for Label-Free Analysis of Foodborne Pathogens and Their Colonization <i>In Vivo</i>

Abstract

Foodborne pathogen infection is a key issue of food safety. Herein, we developed a label-free assay for <i>Salmonella enterica</i> (<i>S. enterica</i>) detection based on the G-quadruplex-probing CRISPR-Cas12 system (termed G-CRISPR-Cas), allowing highly sensitive detection of <i>S. enterica</i> and investigation of their colonization in chickens. The introduction of the G-quadruplex probe serving as the substrate of Cas 12a realized a label-free analysis for foodborne pathogens. Due to the amplification process induced by loop-mediated isothermal amplification (LAMP), G-CRISPR-Cas assay can detect <i>S. enterica</i> as low as 20 CFU. Specificity for pathogenic gene detection was guaranteed by the dual recognition process via LAMP primers and Cas 12a-guided RNA binding. The G-CRISPR-Cas assay was applied to explore <i>S. enterica</i> colonization in the intestinal tract and organs of chickens and showed the risk of <i>S. enterica</i> infection outside of the intestinal tract. The G-CRISPR-Cas assay is promising for on-site diagnosis of the infection or contamination of foodborne pathogens outside the laboratories, such as abattoirs and markets.