The establishment of a duplex real-time PCR assay for rapid and simultaneous detection of bla(NDM) and bla(KPC) genes in bacteria

The latest threat of multidrug-resistant Gram-negative bacteria corresponds to the emergence of carbapenemase New Delhi metallo-β-lactamase (NDM) and Klebsiella pneumoniae carbapenemase (KPC) producers. Rapid molecular detection is essential to limit their spread. In this study, a duplex real-time polymerase chain reaction (PCR) that was specific for the detection of bla(NDM) and bla(KPC) with the same limit of detection of ten plasmid copies was developed. The assay was linear over eight log dilutions for bla(NDM) (R(2) = 0.971; slope, -3.273) and bla(KPC) (R(2) = 0.992; slope, -2.997) with efficiencies of 102% and 115%, respectively. The assay was validated with 157 clinical isolates and showed 100% concordance with conventional PCR. The excellent performance of the duplex PCR assay makes it a powerful tool for surveillance of the carbapenemases NDM and KPC.