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

Abstract

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.

Keywords: Aptamer; Bioassay; DNAzyme; In vitro diagnostic; Methicillin resistant staphylococcus aureus.

Figure 1

a) DNA library and related oligonucleotide sequences used in in vitro selection. b) Schematic illustration of in vitro selection. c) Images of the selection monitored through the fluorescence signal on the beads in each round of positive selection. d) Fluorescein labeled truncated shorter aptamer sequence. e) Binding sensitivity test using dot blot assay in buffer with pure PBP2a. f) Specificity test in buffer using dot blot assay.

Figure 2

Whole cell binding test. a) MRSA33591 was used to test binding with all 6 aptamers. Only PBP2a‐Apt1T1 bound strongly and efficiently. b) Selectivity test with PBP2a‐Apt1T1 against various Stapholococcus species. SL = S. lentus; SC = S. chromogenes; SS = S. saprophyticus; SP = S. pasteuri; SE = S. epidermis. c) Binding test with other aptamers and non‐aptamer sequences. S. pasteuri was found to bind with any DNA sequence. d) LoD for aptamer binding to intact MRSA cells.

Figure 3

a) Schematic of the combined assay using aptamer‐based pulldown of MRSA followed by RCD cleavage and fluorescence signalling. b) Selectivity of MRSA detection relative to MSSA and various non‐target cells, including E. coli, B. subtilis, S. epidermis and S. pasteuri. c) Fluorescence response for varying concentrations of MRSA present in selection buffer. d) Concentration‐response plot for MRSA detection based on fluorescence response at 90 min.

Figure 4

Combined aptamer pull‐down / RCD lateral flow assays of bacteria. a) Selectivity of LFD for MRSA detection relative to MSSA and various non‐target cells, including E. coli, B. subtilis, S. epidermis and S. pasteuri. LFD test line intensity for varying concentrations of MRSA present in: b) selection buffer; c) simulated nasal mucus; d) human serum. Error bars are 1 standard deviation for experiments done in triplicate.