An electrochemical biosensor for the highly sensitive detection of Staphylococcus aureus based on SRCA-CRISPR/Cas12a

Abstract

As one of the major foodborne pathogens, Staphylococcus aureus (S. aureus) can cause infectious diseases. In the current study, a novel electrochemical biosensor based on saltatory rolling circle amplification (SRCA) combined with CRISPR/Cas12a system was developed for the accurate detection of S. aureus. The thio-modified reporter probes (SH-ssDNA-MB) was immobilized on the surface of gold nanoparticle-modified electrode through the Au-S bond. In the presence of S. aureus, the target DNA double strands obtained by SRCA can be specifically recognized with Cas12a/crRNA complex. The trans-cleavage activity of Cas12a induces SH-ssDNA-MB to be cleaved from the electrode surface, resulting in a decrease in the current signal. Subsequently, the ratio of the current can be calculated as the detection result. Under optimal conditions, the detection limits were 2.51 fg/μL for genomic DNA and 3 CFU/mL for S. aureus in pure cultures, respectively. Moreover, the method demonstrated satisfactory specificity, acceptable stability and reproducibility. In comparison with ISO methods, the sensitivity, specificity and accuracy of the developed method were 100%, 97.8% and 98%, respectively. In conclusion, the developed novel electrochemical biosensor provides a potential powerful platform for the accurate detection of S. aureus.

Keywords: CRISPR/Cas12a; Electrochemical biosensor; Saltatory rolling circle amplification; Staphylococcus aureus.