Investigating enzyme kinetics and fluorescence sensing strategy of CRISPR/Cas12a for foodborne pathogenic bacteria.

Foodborne pathogenic bacteria are widespread in various foods, whose cross-contamination and re-contamination are critical influences on food safety. Rapid, accurate, and sensitive detection of foodborne pathogenic bacteria remains a topic of concern. CRISPR/Cas12a can recognize double-stranded DNA directly, showing great potential in nucleic acid detection. However, few studies have investigated the cleavage properties of CRISPR/Cas12a. In this study, the trans -cleavage properties of LbCas12a and AsCas12a were investigated to construct the detection methods for foodborne pathogenic bacteria. The highly sensitive fluorescent strategies for foodborne pathogens were constructed by analyzing the cleavage rates and properties of substrates at different substrate concentrations. Cas12a was activated in the presence of foodborne pathogenic target sequence was present, resulting in the cleavage of a single-stranded reporter ssDNA co-labelled by fluorescein quencher and fluorescein. The sensitivity and specificity of the Cas12a fluorescent strategy was investigated with Salmonella and Staphylococcus aureus as examples. The results showed that AsCas12a was slightly more capable of trans -cleavage than LbCas12a. The detection limits of AsCas12a for Salmonella and Staphylococcus aureus were 24.9 CFU mL−1 and 1.50 CFU mL−1, respectively. In all the seven bacteria, Staphylococcus aureus and Salmonella were accurately discriminated. The study provided a basis for constructing and improving the CRISPR/Cas12a fluorescence strategies. The AsCas12a-based detection strategy is expected to be a promising method for field detection. [Display omitted] • The trans -cleavage ability of LbCas12a and AsCas12a was compared. • AsCas12a showed better affinity and greater reaction rate with trans -cleaved substrates. • CRISPR/Cas12a accurately distinguished Salmonella and Staphylococcus aureus from five other food-borne pathogens. • AsCas12a fluorescence sensing strategy with good detection performance in food matrices. [ABSTRACT FROM AUTHOR]