title: Rapid and sensitive detection of antibiotic resistance on a programmable digital microfluidic platform.
abstract: The widespread dissemination of CTX-M extended spectrum β-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.
authors.affiliation: Electronics and Computer Science, University of Southampton, Southampton, SO17 1BJ, UK. hm@ecs.soton.ac.uk.
authors.forename: Sumit
authors.initials: S
authors.lastname: Kalsi
authors.forename: Martha
authors.initials: M
authors.lastname: Valiadi
authors.forename: Maria-Nefeli
authors.initials: MN
authors.lastname: Tsaloglou
authors.forename: Lesley
authors.initials: L
authors.lastname: Parry-Jones
authors.forename: Adrian
authors.initials: A
authors.lastname: Jacobs
authors.forename: Rob
authors.initials: R
authors.lastname: Watson
authors.forename: Carrie
authors.initials: C
authors.lastname: Turner
authors.forename: Robert
authors.initials: R
authors.lastname: Amos
authors.forename: Ben
authors.initials: B
authors.lastname: Hadwen
authors.forename: Jonathan
authors.initials: J
authors.lastname: Buse
authors.forename: Chris
authors.initials: C
authors.lastname: Brown
authors.forename: Mark
authors.initials: M
authors.lastname: Sutton
authors.forename: Hywel
authors.initials: H
authors.lastname: Morgan
mesh_terms: D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics*; D004352:Drug Resistance, Microbial / Q000235:genetics*; D004566:Electrodes; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics*; D005453:Fluorescence; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation; D010316:Particle Size; D013499:Surface Properties; D013696:Temperature; D013997:Time Factors
chemical_list: D004269:DNA, Bacterial