A Multiplex Recombinase-Aided qPCR Assay for Highly Sensitive and Rapid Detection of khe, bla_KPC-2, and bla_NDM-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.