Development of a DNA biosensor to detect antimicrobial resistance

  • Adrian Butterworth

Student thesis: Doctoral Thesis

Abstract

Antimicrobial resistance is a growing worldwide healthcare challenge, with increasing resistance rates limiting treatment options for common bacterial infections. Advances in diagnostic procedures can provide treatment options more rapidly, improving patient outcomes and maintaining the efficacy of our current antibiotic stockpiles. In this thesis, the development of a DNA biosensor for antibiotic resistance is detailed. Based on electrochemical techniques, the binding of resistance gene sequences at probe modified electrode surfaces is detected. Different low-cost electrode formats are examined for their suitability as DNA sensing platforms. Detection is based upon the formation of a self-assembled monolayer consisting of blocking and probe molecules, and different methods for forming these layers are also explored. Amplification reactions are designed to enrich the specific DNA target using both polymerase chain reaction and isothermal techniques. Amplicons from these reactions are tested on different carbon and gold electrode substrates, as well as on a low-cost potentiostat system which could provide a cost effective, self-contained diagnostic platform. DNA targets are detected on each of these systems, and performance of the low-cost potentiostat is shown to be comparable to a benchtop potentiostat. A solid-phase amplification reaction is then developed, which is used to attach a horseradish peroxidase enzyme label directly to the DNA amplicon produced at the electrode surface. This process is used to detect cells containing antimicrobial resistance gene sequences from culture medium using a simple heat lysis as preparation. Cultures of resistance bearing E. coli are distinguished from blank and S. aureus templates using this solid-phase system, with a limit of detection of 319 CFU/mL detected in under one hour. This system shows promise for further development of cost-effective genetic sensing for rapid identification of antimicrobial resistance genes.
Date of Award31 Aug 2021
Original languageEnglish
Awarding Institution
  • University Of Strathclyde
SponsorsUniversity of Strathclyde
SupervisorDamion Corrigan (Supervisor) & Mary Grant (Supervisor)

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