Fast and sensitive isothermal DNA assay using micro-bead dielectrophoresis for detection of anti-microbial resistance genes

Abstract

Antimicrobial resistant pathogens are a growing worldwide threat to human health. This study proposes a novel method for rapid and sensitive detection of antimicrobial resistance (AMR) genes, specifically blaCTX-M-15 which encodes for the enzyme that offers resistance to extended spectrum -lactam antibiotics. The method combines isothermal DNA amplification by recombinase polymerase amplification (RPA), with microbead dielectrophoresis (DEP)-based DNA detection. The amplicon of RPA is attached to dielectric microbeads, and the amount of amplicon determined by dielectrophoretic impedance measurement (DEPIM) of the microbeads. Amplicon-labeled microbeads were prepared by two-step and one-step labeling. A purified recombinant plasmid containing blaCTX-M-15 and genomic DNA (with plasmid) extracted from AMR bacteria (Escherichia coli NCTC 13441) were used as target samples. A one-step method in which RPA and DNA immobilization on the microbeads are carried out simultaneously, has a detection limit of 2 copies/reaction for the pure plasmid and 50 copies/reaction for genomic DNA. The assays are quantitative with a dynamic range up to 105 copies/reaction, with a total detection time of 26 min. Both methods are easy, rapid, and unlike lateral flow detection are quantitative.

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ORCID for Hywel Morgan: orcid.org/0000-0003-4850-5676

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