Solid-phase helicase dependent amplification and electrochemical detection of Salmonella on highly stable oligonucleotide-modified ITO electrodes

Abstract

An on-surface isothermal helicase-dependent amplification is devised for simple, point-of-need quantification of bacterial genomes. The method relies on the enzyme-extension of a thiol-modified reverse primer anchored to indium tin oxide electrodes, which shows strikingly high thermal and storage stability. Amplification and electrochemical detection of only 10 genomes are thus performed on the same platform without thermal cycling.

On‐Gold Recombinase Polymerase Primer Elongation for Electrochemical Detection of Bacterial Genome: Mechanism Insights and Influencing Factors

“…As modifications in the reaction time and the deployment of dynamic incubation (movement of the sensing chip) were fruitless for improving reliability of the assay at low target concentrations, we evaluated the influence of incorporating both forward and reverse primers in the RPA solution. As previously reported for RPA and other isothermal amplification techniques, small amounts of DNA can be reliably detected as a consequence of an enhanced amplification efficiency when both members of the primer pair are present in the solution. On‐surface RPA amplification of 1 pM T 86 as well as the corresponding negative template control were therefore conducted by introducing 6‐FAM‐FP 2 and RP in solution at 480 nM.…”

“…have demonstrated via a PCR‐based assay the reliability of the regulatory (therefore, highly conserved) bip A (or typ A) gene for the specific detection of Salmonella spp., including the Salmonella serovars undetected by inv A‐based PCR methods . Then, the primer pair described for PCR amplification of an 86 bp DNA fragment specific of pathogenic Salmonella was successfully adapted with minor modifications for solid‐phase helicase dependent amplification (HDA) by our group . Herein we propose the use of this set of primers suited for HDA to gain more insights into the on‐solid recombinase polymerase amplification (on‐solid RPA).…”

Isothermal Nucleic Acid Amplification Techniques and Their Use in Bioanalysis

“…For example, HDA in combination with the lateral flow test was used by Tang et al [31] for identification of Salmonella typhimurium in water samples with the detec tion limit of 100 CFU/ml. HDA with electrochemical signal detection was used for the quantitative determination of Salmonella species [32]. The capture probe was immobilized on the electrode surface, while the signaling primer was modified with fluorescein.…”

A review of microfabricated electrochemical biosensors for DNA detection

“…They then demonstrate a combination of electrochemical and optical measurement techniques by characterizing DNA hybridization on a silane anchored SAM, using Fourier Transform Infra-Red Spectroscopy at the same time as cyclic voltammetry. The advantages of ITO are also utilized by Barreda-Garcia et al, who developed an optimized biosensor for detecting Salmonella (Barreda- García et al, 2017García et al, , 2018. They demonstrate isothermal gene amplification on the same surface as their sensor and were able to detect single molecules of the target.…”