Simultaneous voltammetric determination of E. coli and S. typhimurium based on target recycling amplification using self-assembled hairpin probes on a gold electrode

Abstract

The authors report on a rapid voltammetric method for simultaneous determination of the pathogens E. coli and Salmonella typhimurium (S. typh.) by detecting the rfbE gene of E. coli O157:H7 and gyrB gene of S. typh., respectively, and by using polymerase-assisted target recycling amplification. The assay was constructed by self-assembly of the respective hairpin probes (labeled with the electrochemical probes Methylene Blue and ferrocene) on the surface of a gold electrode. After hybridization between target DNA and hairpin probes (HPs) has occurred, the primers hybridize with the open-chain HPs and initiate extension reactions in the presence of polymerase and deoxyribonucleoside triphosphates. This results in the release of the redox labels from the electrode surface and the target dissociating from the HPs. The released target will bind to other HPs to activate new cycles, which results in enhanced suppression of current, measured best at −0.27 V and +0.36 V (vs. Ag/AgCl) for parallel detection of E. coli DNA and S. typh. DNA, respectively. The method presented here based on target recycling amplification and its integration into multiplexed electrochemical detection of pathogens was successfully applied to quantitative determination of E. coli O157:H7 and S. typh. in synthetic samples. In our perception, the strategy presented here represents a rapid and universal platform for sensitive and multiplexed quantitation of pathogens and related molecular diagnostic targets of relevance in food safety control.