CRISPR/Cas9 bridged recombinase polymerase amplification with lateral flow biosensor removing potential primer-dimer interference for robust Staphylococcus aureus assay

Bloodstream infections have become a major global public health problem with high mortality. Rapid and robust pathogen diagnostics are necessary for improving patient care. Herein, we report an assay for the detection of Staphylococcus aureus (S. aureus) based on the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and recombinase polymerase amplification (RPA) integrated lateral flow biosensor (LFB), namely CCR-LFB. Unlike existing LFBs based on dual-labeled amplicon bridging, CCR-LFB uses CRISPR/Cas9-mediated specific recognition of the amplicons induced by S. aureus DNA to form complexes that bind gold nanoparticles (GNPs) to the test (T) line. This rational design can completely overcome primer-dimer interference without reducing amplification efficiency, thereby improving sensitivity and robustness. Under optimal experimental conditions, S. aureus as low as 102 CFU/mL can be visually judged and a detection limit of 63 CFU/mL was achieved by ImageJ analysis. Additionally, this method has been successfully used to detect S. aureus in real blood samples without any cross-reaction. We hope that this method can serve as a model tool to meet the growing need for point-of-care diagnosis of bloodstream infections.