Ultra-Sensitive and Rapid Detection of Pathogenic Yersinia enterocolitica Based on the CRISPR/Cas12a Nucleic Acid Identification Platform

Abstract

Yersinia enterocolitica is a dangerous foodborne human pathogen that mainly causes gastroenteritis. Ideal methods for the detection of pathogens in food should be rapid, sensitive, specific, and cost effective. To this end, novel in vitro nucleic acid identification methods based on clustered, regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas) endonuclease have received increasing attention. In this study, a simple, visual, and ultrasensitive method, based on CRISPR/Cas12a with recombinase polymerase amplification (RPA), was developed for the detection of Y. enterocolitica. The results show that a specific attachment invasion locus gene (ail) can be rapidly detected using a CRISPR/Cas12a-RPA-based system. Application of the method to raw pork, which was artificially infected with Y. enterocolitica, achieved an estimated detection limit of 1.7 CFU/mL in less than 45 min, and this was 100 times lower compared with qPCR. The results indicated that the CRISPR/Cas12a-RPA system has good potential for monitoring pathogenic Y. enterocolitica in the chilled meat supply chain.