Detection of KPC-Producing Carbapenem-Resistant Klebsiella pneumoniae Based on CRISPR Cas12a
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To develop a detection system for Klebsiella pneumoniae carbapenemase (KPC) and provide a reference for clinical prevention and control of nosocomial infections caused by multidrug-resistant K. pneumoniae. 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%, Kappa 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 K. pneumoniae (CRKP) demonstrates high sensitivity, specificity, and broad applicability. This method requires standard molecular biology equipment but does not rely on sequencing-based platforms.
Abstract
To develop a detection system for Klebsiella pneumoniae carbapenemase (KPC) and provide a reference for clinical prevention and control of nosocomial infections caused by multidrug-resistant K. pneumoniae. 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%, Kappa 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 K. pneumoniae (CRKP) demonstrates high sensitivity, specificity, and broad applicability. This method requires standard molecular biology equipment but does not rely on sequencing-based platforms.