INTRODUCTION: The rise of Klebsiella pneumoniae (KP) and carbapenem-resistant KP (CRKP) poses grave threats to public health and food safety, creating an urgent demand for rapid point-of-care testing (POCT). Traditional detection methods are limited by laboratory barriers, making them unsuitable for POCT implementation.
METHODS: Herein, a one-tube assay integrating recombinant polymerase amplification (RPA) with CRISPR/Cas12a technology was developed for the rapid, sensitive and specific detection of KP and-carrying CRKP. Specific primers targeting the KP-specific rpoB gene and carbapenem-resistance genewere designed, and optimal primer pairs were screened via agarose gel electrophoresis. CrRNA sequences were designed according to RPA amplicons, and the components of the CRISPR/Cas12a reaction were optimized. A two-step reaction system was initially evaluated, followed by the establishment of an integrated one-tube RPA-CRISPR/Cas12a assay. A total of 66 clinical specimens and artificially contaminated food samples were used for method validation, with microbial culture and qPCR as reference methods. blaOXA-48blaOXA-48
RESULTS: The two-step assay was capable of detecting bacterial suspensions at a concentration of 100 CFU/mL. The one-tube system could be completed within 1 hour at 37 °C. This assay avoided aerosol contamination and allowed visual result readout under blue light. In the validation test, the detection results of the one-tube assay were consistent with those obtained by microbial culture and qPCR.
DISCUSSION: This study constructed a dual-target RPA-CRISPR/Cas12a platform for the visual detection of KP and-positive CRKP under blue light. This assay reduces reliance on sophisticated equipment and professional personnel. It can serve as a promising POCT tool for clinical diagnosis and food safety surveillance, and provides evidence for the timely formulation of rational antimicrobial treatment strategies. blaOXA-48
