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 bla KPC , 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 bla KPC and achieved a lower limit of detection of approximately 2.5 copies/μL by repeatedly amplifying a recombinant plasmid containing bla KPC . The RPA-Cas13a assay demonstrated a sensitivity of 96.5% and specificity of 100% when tested on 57 bla KPC -positive CRE strains, which were confirmed by DNA sequencing. Moreover, in 311 sputum samples, the theoretical antibiotic resistance characteristics of bla KPC -positive strains obtained by the RPA-Cas13a assay were highly consistent with the results of antibiotic susceptibility test ( Kappa = 0.978 > 0.81, P