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Simultaneous enzyme-based media can be used to identify both coliforms and E. coli faster than lactose-based fermentation protocols. However, these media can be prone to false-positive results and would require verification tests to confirm the presence of E. coli. These verification tests (e.g. indole or oxidase tests) work based on identifying specific phenotypic traits in bacteria, and hence are also prone to inaccuracies. This study aimed to achieve rapid verification test of E. coli using DNAzyme-based biosensor. The biosensor was first tested for four E. coli strains against more than 90 non-E. coli strains routinely causing false positive results and showed high specificity towards E. coli strains. The biosensor was then determined for its detection sensitivity. It was observed that a single E. coli colony would generate a robust fluorescence signal after 2 h of sub-culturing. With this short incubation time, non-E. coli strains were unable to generate any apparent fluorescence signal, hence further improving the detection specificity of biosensor. Lastly, we further demonstrate that the biosensor can be applied to different commercial brands of simultaneous enzyme-based agar to consistently identify the presence of E. coli. The biosensor was able to rapidly denote presence of discrete E. coli colonies by binding on to protein targets possibly of DNA replication or motility functions that were predominantly specific to E. coli.

DNAzyme-based biosensor as a rapid and accurate verification tool to complement simultaneous enzyme-based media forE. colidetection