CRISPR/Cas13-assisted carbapenem-resistant Klebsiella pneumoniae detection

Carbapenem-resistant Klebsiella pneumoniae (CRKP) is capable of causing serious community and hospital-acquired infections. However, currently, the identification of CRKP is complex and inefficient. Hence, this study aimed to develop methods for the early and effective identification of CRKP to allow reasonable antimicrobial therapy in a timely manner.

K. pneumoniae (KP)-, K. pneumoniae carbapenemase (KPC)- and New Delhi metallo-β-lactamase (NDM)- specific CRISPR RNAs (crRNAs), polymerase chain reaction (PCR) primers and recombinase-aided amplification (RAA) primers were designed and screened in conserved sequence regions. We established fluorescence and lateral flow strip assays based on CRISPR/Cas13a combined with PCR and RAA, respectively, to assist in the detection of CRKP. Sixty-one clinical strains (including 51 CRKP strains and 10 carbapenem-sensitive strains) were collected for clinical validation.

Using the PCR-CRISPR assay, the limit of detection (LOD) for KP and the blaKPC and blaNDM genes reached 1 copy/μL with the fluorescence signal readout. Using the RAA-CRISPR assay, the LOD could reach 10¹ copies/μL with both the fluorescence signal readout and the lateral flow strip readout. Additionally, the positivity rates of CRKP-positive samples detected by the PCR/RAA-CRISPR fluorescence and RAA-CRISPR lateral flow strip methods was 92.16% (47/51). The sensitivity and specificity reached 100% for KP and blaKPC and blaNDM gene detection. For detection in a simulated environmental sample, 1 CFU/cm² KP could be detected.

We established PCR/RAA-CRISPR assays for the detection of blaKPC and blaNDM carbapenemase genes, as well as KP, to facilitate the detection of CRKP.