To establish a rapid molecular detection method for Klebsiella pneumoniae carbapenemase (KPC) carbapenemase (bla_KPC) genes based on recombinase aided amplification (RAA)-clusteredregularly interspaced short palindromic repeats (CRISPR)-CRISPR associated (Cas)12a (CRISPR-Cas12a) technology.

Specific RAA primers and CRISPR RNA(crRNA) were designed targeting the bla_KPC gene sequence to construct a RAA-CRISPR-Cas12a detection assay. 4 strains of Klebsiella pneumoniae carrying the bla_KPC gene stored in Lecong Hospital of Shunde, Foshan from 2022 to 2023 were collected for method research, and 40 clinical strains of Klebsiella pneumoniae for method evaluation. At the same time, quantitative real-time polymerase chain reactio (qPCR) method was performed in parallel to compare detection rates and concordance between the two methods.

The RAA-CRISPR-Cas12a method achieved a sensitivity of 10 copies/µl for bla_KPC gene detection. At the same time, RAA-CRISPR-Cas12a and qPCR methods were used to detect 40 clinical strains of Klebsiella pneumoniae. Both methods simultaneously detected 5 bla_KPC-positive and 35 bla_KPC-negative isolates. Using qPCR as the gold standard, the sensitivity of the method established in this study was 100% (5/5), and the sensitivity was 100% (35/35), with a 100% agreement rate between the two methods.

The established RAA-CRISPR-Cas12a method provides a reliable and efficient tool for bla_KPC gene detection, facilitating rapid clinical screening of bla_KPC gene.