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

Fast and sensitive isothermal DNA assay using micro-bead dielectrophoresis for detection of anti-microbial resistance genes
Fast and sensitive isothermal DNA assay using micro-bead dielectrophoresis for detection of anti-microbial resistance genes
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.
0956-5663
583-589
Nakano, MIchihiko
fbbd1e17-7f3c-4d33-a716-18fddcdb7192
Kalsi, Sumit
0ec1f5c2-a6ec-4c07-92fb-15708c28742e
Morgan, Hywel
de00d59f-a5a2-48c4-a99a-1d5dd7854174
Nakano, MIchihiko
fbbd1e17-7f3c-4d33-a716-18fddcdb7192
Kalsi, Sumit
0ec1f5c2-a6ec-4c07-92fb-15708c28742e
Morgan, Hywel
de00d59f-a5a2-48c4-a99a-1d5dd7854174

Nakano, MIchihiko, Kalsi, Sumit and Morgan, Hywel (2018) Fast and sensitive isothermal DNA assay using micro-bead dielectrophoresis for detection of anti-microbial resistance genes. Biosensors & Bioelectronics, 117, 583-589. (doi:10.1016/j.bios.2018.06.063).

Record type: Article

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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More information

Submitted date: 30 May 2018
Accepted/In Press date: 28 June 2018
e-pub ahead of print date: 30 June 2018
Published date: 15 October 2018

Identifiers

Local EPrints ID: 422210
URI: https://eprints.soton.ac.uk/id/eprint/422210
ISSN: 0956-5663
PURE UUID: 9847f931-f2b1-4b20-a9eb-3514381b3727
ORCID for Hywel Morgan: ORCID iD orcid.org/0000-0003-4850-5676

Catalogue record

Date deposited: 18 Jul 2018 16:31
Last modified: 15 Oct 2019 00:45

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