Sensitive detection of foodborne pathogens based on CRISPR‐Cas13a

Abstract

Salmonella, being one of the most widespread foodborne pathogens, is a compulsory test item required by national food safety standard of China and many other countries. More sensitive and specific Salmonella detection method is still needed since traditional methods are time consuming and highly dependent on enormous manpower and material resources. In this research, a bacteria detection method based on CRISPR‐Cas13a system (where CRISPR is Clustered Regularly Interspaced Short Palindromic Repeats) was proposed. The target DNA was amplified by PCR and transcribed into RNA by T7 transcriptase, which can activate the RNase activity of the Cas13a protein. The self‐folding quenched fluorescent probe can be cleaved by the activated Cas13a protein to generate fluorescent signal. We named this method as PCF detection (PCR‐CRISPR‐Fluorescence based nucleic acid detection). In this study, PCF detection showed excellent sensitivity, which can detect Salmonella genomic DNA with a minimum of 101 aM or 10° CFU/ml Salmonella bacteria in 2 hr. It also showed good specificity with no cross‐reaction with other common foodborne bacteria. Practical Application The PCF detection method proposed in this article can detect Salmonella sensitively and specifically, providing a novel strategy for the detection of foodborne pathogens in food and has great application potential in other microbial detection fields.

CRISPR‐Cas‐based detection for food safety problems: Current status, challenges, and opportunities

“…CRISPR-Cas13a could be used as an ideal detection tool. CRISPR-Cas13 can show the same collateral cleavage as Cas12, but the recognized target and nonspecific cleaved single strand are RNA (Gao et al, 2021). Zhang, Zhou, et al (2021) constructed a CRISPR-Cas13a assay, which was capable of achieving mix-andread detection of viable pathogenic bacteria.…”

“…CRISPR‐Cas13a could be used as an ideal detection tool. CRISPR‐Cas13 can show the same collateral cleavage as Cas12, but the recognized target and nonspecific cleaved single strand are RNA (Gao et al., 2021). Zhang, Zhou, et al.…”

Recent advances in CRISPR‐based systems for the detection of foodborne pathogens

“…Experiments by J. Zhou et al (2020) and S. Gao et al (2021) both took advantage of Cas13a, to detect Salmonella and S. aureus DNA, respectively. Both their methods involved transcribing the DNA into RNA to apply the Cas13a effector protein.…”

Recent Advances in Electrochemical Biosensors for the Detection of Salmonellosis: Current Prospective and Challenges

“…Song et al transcribed the target DNA into RNA T7 transcriptase and stimulated the RNase activity of the Cas13a protein. The active Cas13a protein cleaved the self-folding quenched fluorescence probe to generate the fluorescent signal [ 113 ]. The integration of CRISPR/Cas technology in electrochemical sensors would help in the detection of pathogens at a genomic level.…”