Graphene oxide assisting the visual detection of Salmonella by CRISPR/Cas12a

Abstract

Rapid and accurate detection of Salmonella is extremely important to ensure food safety. Combining nucleic acid amplification with CRISPR/Cas12a can realize sensitive and specific detection of foodborne pathogens. However, present CRISPR/Cas-based methods mainly depend on fluorophore and quencher dual-labeled ssDNA as the reporter, which brings in high cost and steric hindrance. Herein, a new signal output format has been developed. The method takes advantage of graphene oxide adsorbing FAM-ssDNA and quenching the fluorescent emission while CRISPR/Cas12a recovering the fluorescent emission by trans-cleaving the FAM-ssDNA. Factors affecting the performance of the sensor have been optimized, and it is shown that the oligonucleotide of 12 nt with the fluorophore modified at the 3′ end displays the highest signal to noise ratio. The signal output method has been combined with recombinase polymerase amplification for detection of Salmonella and presents consistent specificity and sensitivity (5 × 101 copies) as real-time PCR. By replacing the dual-labeled ssDNA reporter with FAM-ssDNA and GO, this signal output method saves ∼65% cost. Notably, this method does not require a real-time thermal cycler or any other sophisticated instrument. Besides pathogens, this easy and convenient method can also be extended to detect other targets.