Foodborne pathogens pose a serious risk to human health, and the simple and rapid detection of such bacteria in complex food matrices remains challenging. Herein, we present the selection and characterization of a novel RNA-cleaving fluorogenic DNAzyme, named RFD-BC1, with exceptional specificity for Burkholderia gladioli pv. cocovenenans (B. cocovenenans), a pathogen strongly associated with fatal food poisoning cases. RFD-BC1 was activated by a protein secreted specifically by whole viable B. cocovenenans and displayed an optimum pH distinct from the selection pH, with a rate constant of approximately 0.01 min-1 at pH 5.0. Leveraging this newly discovered DNAzyme, we developed a novel system, termed DNAzymes-in-droplets (DID), that integrates droplet microfluidics to achieve the rapid and selective detection of live B. cocovenenans with single-cell sensitivity. We believe that the approach described herein holds promise for combating specific bacterial pathogens in food samples, offering significant potential for broader applications in food safety and public health.
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