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Rapid and sensitive detection of antibiotic resistance on a programmable digital microfluidic platform

Rapid and sensitive detection of antibiotic resistance on a programmable digital microfluidic platform
Rapid and sensitive detection of antibiotic resistance on a programmable digital microfluidic platform
The widespread dissemination of CTX-M extended spectrum ?-lactamases among Escherichia coli bacteria, both in nosocomial and community environments, is a challenge for diagnostic bacteriology laboratories. We describe a rapid and sensitive detection system for analysis of DNA containing the blaCTX-M-15 gene using isothermal DNA amplification by recombinase polymerase amplification (RPA) on a digital microfluidic platform; active matrix electrowetting-on-dielectric (AM-EWOD). The devices have 16 800 electrodes that can be independently controlled to perform multiple and simultaneous droplet operations. The device includes an in-built impedance sensor for real time droplet position and size detection, an on-chip thermistor for temperature sensing and an integrated heater for regulating the droplet temperature. Automatic dispensing of droplets (45 nL) from reservoir electrodes is demonstrated with a coefficient of variation (CV) in volume of approximately 2%. The RPA reaction is monitored in real-time using exonuclease fluorescent probes. Continuous mixing of droplets during DNA amplification significantly improves target DNA detection by at least 100 times compared to a benchtop assay, enabling the detection of target DNA over four-order-of-magnitude with a limit of detection of a single copy within ~15 minutes
1473-0197
3065-3075
Kalsi, Sumit
65cb3d4c-c28c-490a-85a3-7a8fb96ecfee
Valiadi, Martha
e245e9c5-3e97-4896-b910-c4178c5fb3c9
Tsaloglou, Maria-Nefeli
99ab30ba-15da-4d25-86ba-608d127f8369
Parry-Jones, Lesley
40b6ef5c-94f7-4a1b-b16f-6c1b2c85e8da
Jacobs, Adrian
cb9acb55-a8b0-4708-aecf-2e39b81d88a6
Watson, Rob
c54ba509-bbf2-4100-a4d2-aac714af26b6
Turner, Carrie
92e6c17b-41e4-4867-9812-677e4cb88996
Amos, Robert
42b7d3bf-4963-406c-a694-bfc313fe2cb2
Hadwen, Ben
5e5414ed-d4b6-49fd-9ad8-1176be50df23
Buse, Jonathan
60bcec3f-8847-4d13-bd64-4464884f0b8e
Brown, Chris
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Sutton, Mark
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Morgan, Hywel
de00d59f-a5a2-48c4-a99a-1d5dd7854174
Kalsi, Sumit
65cb3d4c-c28c-490a-85a3-7a8fb96ecfee
Valiadi, Martha
e245e9c5-3e97-4896-b910-c4178c5fb3c9
Tsaloglou, Maria-Nefeli
99ab30ba-15da-4d25-86ba-608d127f8369
Parry-Jones, Lesley
40b6ef5c-94f7-4a1b-b16f-6c1b2c85e8da
Jacobs, Adrian
cb9acb55-a8b0-4708-aecf-2e39b81d88a6
Watson, Rob
c54ba509-bbf2-4100-a4d2-aac714af26b6
Turner, Carrie
92e6c17b-41e4-4867-9812-677e4cb88996
Amos, Robert
42b7d3bf-4963-406c-a694-bfc313fe2cb2
Hadwen, Ben
5e5414ed-d4b6-49fd-9ad8-1176be50df23
Buse, Jonathan
60bcec3f-8847-4d13-bd64-4464884f0b8e
Brown, Chris
e0f0cd52-62e7-4a99-85d6-340452b6cc35
Sutton, Mark
3ffaeca5-caf0-4d77-a573-fa4763387149
Morgan, Hywel
de00d59f-a5a2-48c4-a99a-1d5dd7854174

Kalsi, Sumit, Valiadi, Martha, Tsaloglou, Maria-Nefeli, Parry-Jones, Lesley, Jacobs, Adrian, Watson, Rob, Turner, Carrie, Amos, Robert, Hadwen, Ben, Buse, Jonathan, Brown, Chris, Sutton, Mark and Morgan, Hywel (2015) Rapid and sensitive detection of antibiotic resistance on a programmable digital microfluidic platform. Lab on a Chip, 15 (14), 3065-3075. (doi:10.1039/c5lc00462d).

Record type: Article

Abstract

The widespread dissemination of CTX-M extended spectrum ?-lactamases among Escherichia coli bacteria, both in nosocomial and community environments, is a challenge for diagnostic bacteriology laboratories. We describe a rapid and sensitive detection system for analysis of DNA containing the blaCTX-M-15 gene using isothermal DNA amplification by recombinase polymerase amplification (RPA) on a digital microfluidic platform; active matrix electrowetting-on-dielectric (AM-EWOD). The devices have 16 800 electrodes that can be independently controlled to perform multiple and simultaneous droplet operations. The device includes an in-built impedance sensor for real time droplet position and size detection, an on-chip thermistor for temperature sensing and an integrated heater for regulating the droplet temperature. Automatic dispensing of droplets (45 nL) from reservoir electrodes is demonstrated with a coefficient of variation (CV) in volume of approximately 2%. The RPA reaction is monitored in real-time using exonuclease fluorescent probes. Continuous mixing of droplets during DNA amplification significantly improves target DNA detection by at least 100 times compared to a benchtop assay, enabling the detection of target DNA over four-order-of-magnitude with a limit of detection of a single copy within ~15 minutes

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Accepted/In Press date: 10 June 2015
Published date: 18 June 2015
Organisations: Nanoelectronics and Nanotechnology

Identifiers

Local EPrints ID: 380916
URI: https://eprints.soton.ac.uk/id/eprint/380916
ISSN: 1473-0197
PURE UUID: 95ca4521-660a-4900-8db2-55a271e32757
ORCID for Hywel Morgan: ORCID iD orcid.org/0000-0003-4850-5676

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Date deposited: 18 Sep 2015 13:59
Last modified: 15 Aug 2019 00:45

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