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.

Rapid detection methods for foodborne pathogens based on nucleic acid amplification: Recent advances, remaining challenges, and possible opportunities

“…For example, Tian et al (2021) compared the use of RPA-CRISPR/Cas12a and qPCR in detecting L. monocytogenes in milk and found that the sensitivity of RPA-CRISPR/Cas12a was 100 times higher than that of the qPCR method, with a detection limit of 10 CFU/mL, without requiring enrichment. Similarly, Xiao et al (2022) found that RPA-CRISPR/Cas12a outperformed the qPCR method in detecting Y. enterocolitica in pork, as it was able to detect this pathogen at a concentration of 1 cfu/mL, whereas qPCR could only detect it at a concentration of 10 2 cfu/mL. In a separate study, Liu et al (2022a) compared the specificity and sensitivity of RPA-CRISPR/Cas12a and qPCR in detecting Salmonella spp.…”

“…A combination of RPA arrays with CRISPR/Cas12a (RPA-CRISPR/Cas12a) can perform better than or at least comparable to the qPCR method in detecting pathogens in food samples ( Liu et al, 2022a , Tian et al, 2021 , Xiao et al, 2022 ). For example, Tian et al (2021) compared the use of RPA-CRISPR/Cas12a and qPCR in detecting L. monocytogenes in milk and found that the sensitivity of RPA-CRISPR/Cas12a was 100 times higher than that of the qPCR method, with a detection limit of 10 CFU/mL, without requiring enrichment.…”

Development of CRISPR-Mediated Nucleic Acid Detection Technologies and Their Applications in the Livestock Industry

“…Additionally, the RPA-CRISPR/Cas12a strategy has been used for the detection of other foodborne pathogenic bacteria ( Listeria monocytogenes , Yersinia enterocolitica , Staphylococcus aureus , Campylobacter jejuni , Escherichia coli , etc.) in food production animals at attomolar level [ 96 , 97 , 98 , 99 , 100 ].…”