Rapid electrochemical genosensing platform for direct detection of CTX-M-15 ESBL gene.

Abstract

Extended-spectrum β-lactamase (ESBL) producing Enterobacteriaceae, particularly CTX-M-15-harboring strains, present significant diagnostic challenges due to limitations of conventional phenotypic and molecular methods. Here, we report a rapid electrochemical genosensing platform for direct CTX-M-15 detection using gold-electrodeposited screen-printed carbon electrode (SPCE) following solution-phase probe-target hybridization. A 22-mer probe targeting conserved CTX-M-15 regions was designed through sequence alignment and validated by Basic Local Alignment Search Tool (BLAST), primer mapping, and thermodynamic analysis. Probe specificity was confirmed by fluorescence in-situ hybridization (FISH) and fluorescence spectroscopy. Electrochemical characterization and detection were performed using square-wave voltammetry (SWV), enabling discrimination between complementary DNA, denatured DNA, and non-target species. Optimization of probe-to-target ratio, hybridization time, methylene blue volume, and SWV frequency established a 15 min assay with a linear range of 2 pM-20 μM (R2 = 0.98) and a detection limit of 2 pM. The sensor exhibited high specificity toward CTX-M-15 over different bacterial strains. It was further clinically validated using clinical isolate samples, showing complete agreement with PCR and disc diffusion results. These findings demonstrate its potential as a rapid, low-cost platform for AMR diagnostics.

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