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

Antimicrobial resistant pathogens are a growing worldwide threat to human health. This study describes a novel method for rapid and sensitive detection of antimicrobial resistance (AMR) genes, specifically bla(CTX-M-15) which encodes for the enzyme that offers resistance to extended spectrum beta-lactam antibiotics. The method combines isothermal DNA amplification by recombinase polymerase amplification (RPA), with microbead dielectrophoresis (DEP)-based DNA detection. The RPA amplicon is captured onto dielectric microbeads, and the amount of amplicon determined by dielectrophoretic impedance measurement (DEPIM) of the microbeads. Amplicon-labeled microbeads were prepared by either a two-step or one-step method. A purified recombinant plasmid containing bla(CTX-M-15) and genomic DNA (with plasmid) extracted from an AMR bacteria (Escherichia coli NCTC 13441) were used as target samples. A one-step method in which RPA and DNA immobilization on the microbeads is carried out simultaneously, has a detection limit of 2 copies/reaction for pure plasmid and 50 copies/reaction for genomic DNA. The assays are quantitative with a dynamic range up to 10(5) copies/reaction, with a total detection time of 26 min. Both methods are easy, rapid, and unlike lateral flow detection are quantitative.