Determination of bacterial DNA based on catalytic oxidation of cysteine by G-quadruplex DNAzyme generated from asymmetric PCR: Application to the colorimetric detection of Staphylococcus aureus.

A one-step, one-tube colorimetric assay is described for the detection of bacterial double-stranded DNA (dsDNA). It utilizes a G-quadruplex DNAzyme produced by an asymmetric polymerase chain reaction (As-PCR) that catalyzes the oxidation of cysteine to form cystine. This results in the formation of oligonucleotide-modified gold nanoparticles via triplex formation, and eventually in a color change from red to blue that occurs within about 10 mins. This can be measured by ratiometric colorimetric (at 525 and 600 nm). The limit of detection (LOD) for the model analyte (dsDNA of Staphylococcus aureus (S. aureus)) is as low as 0.28 pg per 0.05 mL with a good linear response ranging from 16.0 fg·μL-1 to 1.6 ng·μL-1. This is much lower than previously reported LODs. The assay is highly selective for S. aureus dsDNA over a range of other bacterial DNAs. Conceivably, it provides an attractive alternative tool for rapid detection of bacterial dsDNA as required in pathogen screening in the food industry. Graphical abstract Schematic presentation of a colorimetric assay for bacterial DNA. It is based on the catalytic activity of a G-quadruplex DNAzyme that is formed by an asymmetric PCR involving triplex DNA formation and gold nanoparticle (AuNPs) aggregation.