Detection of KPC-Producing Carbapenem-Resistant <i>Klebsiella pneumoniae</i> Based on CRISPR Cas12a

To develop a detection system for <i>Klebsiella pneumoniae</i> carbapenemase (KPC) and provide a reference for clinical prevention and control of nosocomial infections caused by multidrug-resistant <i>K. pneumoniae</i>. The KPC resistance gene was amplified by PCR. Guided by crRNA, Cas12a specifically identified the resistance gene and activated its trans-cleavage activity. In the detection system, a fluorescence probe was cleaved by activated Cas12a, and the fluorescence signal was measured using a microplate reader. Under optimized conditions, the fluorescence signal appeared within 12 min, peaked at 40 min and completed detection within 60 min. sensitivity: 91.2%, specificity: 84.1%, detection limit: 0.01 ng/μl. The samples were examined by fluorescence-CRISPR Cas12a and PCR. The coincidence rate was 85.9%, <i>Kappa</i> value was 0.8. The ROC curve analysis revealed an AUC of 0.916, with an optimal cutoff value of 1.55, sensitivity of 91.2%, and specificity of 84.1%. The CRISPR Cas12a detection of carbapenem-resistant <i>K. pneumoniae</i> (CRKP) demonstrates high sensitivity, specificity, and broad applicability. This method requires standard molecular biology equipment but does not rely on sequencing-based platforms.