Rapid Identification of Antibiotic-Resistance Mutation in <i>Bordetella pertussis</i> Using a One-Pot Isothermal System

Abstract Rapid and accurate genotyping of Bordetella pertussis (BP) is essential for guiding antimicrobial therapy and epidemiological surveillance. However, existing genotyping methods often involve multistep workflows and postamplification handling, increasing the risk of aerosol contamination and limiting their applicability in resource-limited settings. Here, we present a closed-tube, one-pot strategy that integrates loop-mediated isothermal amplification (LAMP) with an enzyme-free catalytic hairpin assembly (CHA) circuit for the simultaneous detection and genotyping of wild-type BP and the antibiotic-resistance A2047G mutant. In this platform, LAMP provides rapid and efficient isothermal amplification, while the CHA circuit directly converts amplification products into fluorescence signals and enables single-nucleotide discrimination through sequence-specific recognition, thereby improving genotyping accuracy without increasing assay complexity. The integrated assay combines rapid detection, high specificity, and a simple closed-tube workflow with minimal instrumentation requirements, making it well suited for point-of-care testing and applications in resource-limited settings.