Rapid Detection of blaKPC in Carbapenem-Resistant Enterobacterales Based on CRISPR/Cas13a

Klebsiella pneumoniae carbapenemase (KPC) is a crucial enzyme that causes carbapenem resistance in Enterobacterales, and infections by these "superbugs" are extremely challenging to treat. Therefore, there is a pressing need for a rapid and accurate KPC detection test to control the prevalence of carbapenem-resistant Enterobacterales (CREs). In this study, we established a novel method for detection of blaKPC, the gene responsible for encoding KPC, based on a recombinase polymerase amplification (RPA) and a CRISPR/Cas13a reaction coupled to fluorophore activation (termed RPA-Cas13a assay). We carefully selected a pair of optimal amplification primers for blaKPC and achieved a lower limit of detection of approximately 2.5 copies/μL by repeatedly amplifying a recombinant plasmid containing blaKPC. The RPA-Cas13a assay demonstrated a sensitivity of 96.5