A Combined Aptamer Pulldown-DNAzyme Cleavage Assay for Intact Methicillin Resistant Staphylococcus aureus Cells

Accurate and convenient detection of methicillin resistant Staphylococcus aureus (MRSA) plays a vital role in determining appropriate antibiotic interventions. Herein, we report the first example combining a DNA aptamer and an RNA-cleaving DNAzyme (RCD) that bind different protein markers for selective preconcentration and detection of MRSA. An aptamer for the penicillin binding protein 2a (PBP2a) was generated by in vitro selection and was coupled to agarose beads to allow rapid pull down of intact MRSA cells from complex samples. A previously reported RCD for Staphylococcus aureus (SA) was then used to detect a second proteinaceous marker within the lysed cells based on a protein-activated cleavage reaction that was linked to both fluorescence and lateral flow assays, allowing for selective detection of MRSA over methicillin sensitive SA. An optimized LFD assay could detect ∼10³ cfu mL^-1 of MRSA in either nasal mucus or serum with a total assay time of 1 h using minimal sample processing.