A novel specific fluorescent biosensor for detection of foodborne pathogens based on split crRNA of CRISPR/Cas12a

A novel CRISPR/Cas12a biosensor for specific detection of pathogenic bacteria was constructed based on target nucleic acid (NA) inducing transcription-unleashed split crRNA. Two NA sites of bacterial 16S rRNA were bound to specific probes to induce transcription of large amounts of split crRNA, namely crRNA1 and crRNA2. The amplified crRNA1 and crRNA2 formed a complex with Cas12a and single-strand (ss) DNA activators. Then, Cas12a mediated trans cleavage of the ssDNA reporter molecules, which produced a fluorescent signal. Under optimum conditions, the developed fluorescent biosensor for detection of Staphylococcus aureus showed excellent performance with a low detection limit of 21 CFU/mL and a wide linear range of 103-106 CFU/mL. The proposed biosensor was sensitive and specific for detection of Staphylococcus aureus in spiked milk samples.