Duplex Recombinase Polymerase Amplification‐Lateral Flow Assay for Rapid Visual Detection of Critical Antibiotic Resistance Genes in Escherichia coli and Klebsiella pneumoniae From Positive Blood Cultures

Background Bloodstream infections (BSIs) caused by multidrug‐resistant bacteria (MDR) are a leading cause of mortality and morbidity among hospitalized patients. Extended‐Spectrum Beta‐Lactamase (ESBL), carbapenem‐resistant Enterobacterales (CRE), and colistin resistance are critically important antibiotic‐resistant bacteria. The rapid identification of these critical antibiotic resistance genes from positive blood cultures is essential for accelerating the diagnostic turnaround time, enabling early therapy, and supporting effective antibiotic stewardship programs. Methods Recombinase Polymerase Amplification (RPA) integrated with an in‐house Lateral Flow Assay (LFA) for duplex detection of bla CTX‐M / mcr‐1 and bla oxa‐48‐like / bla NDM was established in this study. 5′ modified primers compatible with those immobilized antibodies on LFA were designed. The primer concentrations, temperature, and time were optimized at 0.2–0.6 μM, 36°C–39°C, and 20–30 min, respectively. The duplex RPA amplicons were diluted in buffer and visually detected on in‐house lateral flow strips within 10–15 min. The limit of detection (LOD), specificity, and method validation were evaluated. Results The optimal primer concentrations of bla CTX‐M / mcr‐1 (0.35 μM/0.35 μM) and bla oxa‐48‐like / bla NDM (0.2 μM/0.6 μM) provided balanced signal intensities for all target genes at 37°C–38°C within 25 min. The LOD across incubation time of 0–8 h was determined as 10 7 –10 2 CFU/mL. No cross‐reactivity was observed with other common Gram‐negative pathogens. Sensitivity and specificity of duplex RPA‐LFA for bla CTX‐M , mcr‐1 , and bla oxa‐48‐like / bla NDM detection were 85.71%–98.28% and 93.62%–100%. The accuracy was 93.33%–96.13%. Conclusion This developed duplex RPA‐LFA assay is well‐suited for rapid screening in positive blood cultures, providing a cost‐effective tool for the treatment, surveillance, and control of MDR in critical infections.