Electrochemical Detection of Oxacillin Resistance using Direct-Labeling Solid-Phase Isothermal Amplification.
Adrian ButterworthPratibha PratibhaAndreas MarxDamion K CorriganPublished in: ACS sensors (2021)
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.
Keyphrases
- nucleic acid
- label free
- escherichia coli
- loop mediated isothermal amplification
- real time pcr
- genome wide
- low cost
- gold nanoparticles
- antibiotic resistance genes
- high throughput
- wastewater treatment
- nitric oxide
- genome wide identification
- transcription factor
- circulating tumor
- dna methylation
- hydrogen peroxide
- cell death
- risk assessment
- single molecule
- ionic liquid
- climate change
- cell cycle arrest
- endoplasmic reticulum stress
- genome wide analysis
- cell proliferation
- pi k akt
- anaerobic digestion
- high resolution
- single cell
- klebsiella pneumoniae