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.
Keywords: DNA biosensing; antimicrobial resistance; modified nucleotides; recombinase polymerase amplification; solid-phase amplification.