CRISPR/Cas12a based fluorescence-enhanced lateral flow biosensor for detection of Staphylococcus aureus

Rapid, accurate point-of-care testing (POCT) for pathogenic bacteria detection is the key for avoiding foodborne diseases caused by pathogens or their toxins. The lateral flow biosensor (LFB) based on clustered regularly interspaced short palindromic repeats (CRISPR/Cas) has displayed remarkable potential for pathogen diagnosis. In this study, we report a novel CRISPR/Cas12a-based fluorescence enhanced LFB in conjunction with functionalized quantum dots, combined with recombinase-assisted amplification (RAA), to establish low-cost, simple, and sensitive detection of Staphylococcus aureus, namely CRA-LFB (CRISPR/Cas-recombinase-assisted amplification based LFB). The CRA-LFB assay is characterized by Cas12a-mediated trans-cleavage activation induced by the target DNA to digest biotin-DNA probes, caused no complementarity to the capture probe immobilized on the test (T) line, and resulted in an undetectable T line fluorescence signal on LFB. The naked eye or fluorescence strip reader was used to determine the fluorescence intensity of the T and control lines. The limit of detection (at optimal conditions) was as low as 75 aM of genomic DNA, and 5.4 × 102 cfu/mL of S. aureus in pure cultures were detected. Moreover, this CRA-LFB assay can rapidly and accurately detect S. aureus in spiked and natural meat and vegetable samples. The CRA-LFB method yielded high specificity and no interference from other nontargeted bacteria. The results exhibited high-resolution, high-intensity fluorescent signals obtained within 70 min. This bacterial detection platform is simple, low cost, almost equipment-free, and fully qualified for food and clinical diagnosis onsite testing requirements. [Display omitted] •We developed a novel, fluorescence-enhanced CRISPR/Cas12a integrated LFB for pathogen detection.•Clever design and binding method allowing a antibody-free, low cost and almost equipment-free LFB for POCT.•The special target and CRISPR/Cas12a system based LFB assay enables highly-specific detection of Staphylococcus aureus.•Without enrichment culture, the detection limit of 75 aM is reported within 70 min.•The biosensor shows excellent practical performance in natural food samples.