Electrochemical detection of oxacillin resistance using direct-labelling solid-phase isothermal amplification

Adrian Butterworth, Pratibha Pratibha, Andreas Marx, Damion K. Corrigan

Research output: Contribution to journalArticlepeer-review

Abstract

Isothermal amplification reactions represent an important and exciting approach to achieve widespread, low cost, and easily implemented molecular diagnostics. This work presents a modified recombinase polymerase amplification (RPA) reaction, which can be directly coupled to a simple electrochemical measurement to ultimately allow development of a nucleic acid-based assay for antibiotic resistance genes. It is shown that use of reagents from a standard RPA reaction kit allows incorporation of horse radish peroxidase-labeled thymine nucleotides into amplified DNA strands, which can be detected via an amperometric signal readout for detection of important gene sequences. The assay is exemplified through detection of fragments of the oxacillin resistance gene in Escherichia coli cells bearing a drug resistance plasmid, achieving a potential limit of detection of 319 cfus/mL and an unoptimized time to result of 60 min. This work serves as a suitable demonstration of the potential for a system to deliver detection of key drug resistance genes at clinically relevant levels.
Original languageEnglish
Pages (from-to)3773-3780
Number of pages8
JournalACS Sensors
Volume6
Issue number10
Early online date1 Oct 2021
DOIs
Publication statusPublished - 22 Oct 2021

Keywords

  • peptides and proteins
  • bacteria
  • genetics
  • monomers
  • electrodes
  • DNA biosensing
  • modified nucleotides
  • recombinase polymerase amplification
  • antimicrobial resistance
  • solid-phase amplification

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