A Multiplex Recombinase‐Aided qPCR Assay for Highly Sensitive and Rapid Detection of khe, blaKPC‐2, and blaNDM‐1 Genes in Klebsiella pneumoniae

Objective This study aimed to establish a highly sensitive and rapid single‐tube, two‐stage, multiplex recombinase‐aided qPCR (mRAP) assay to specifically detect the khe, bla KPC‐2 , and bla NDM‐1 genes in Klebsiella pneumoniae . Methods mRAP was carried out in a qPCR instrument within 1 h. The analytical sensitivities of mRAP for khe, bla KPC‐2 , and bla NDM‐1 genes were tested using recombinant plasmids and dilutions of reference strains. A total of 137 clinical isolates and 86 sputum samples were used to validate the clinical performance of mRAP. Results mRAP achieved the sensitivities of 10, 8, and 14 copies/reaction for khe, bla KPC‐2 , and bla NDM‐1 genes, respectively, superior to qPCR. The Kappa value of qPCR and mRAP for detecting khe, bla KPC‐2 , and bla NDM‐1 genes was 1, 0.855, and 1, respectively ( p < 0.05). Conclusion mRAP is a rapid and highly sensitive assay for potential clinical identification of khe, bla KPC‐2 , and bla NDM‐1 genes in K. pneumoniae .