Real-time PCR assay and a synthetic positive control for the rapid and sensitive detection of the emerging resistance gene New Delhi Metallo-β-lactamase-1 (bla _NDM-1)

Abstract

Carbapenems are important last-line antibiotics for the treatment of hospital infections. Enterobacteriaceae (such as Klebsiella pneumoniae or Escherichia coli) expressing the “New Delhi Metallo-β-lactamase” gene bla _NDM-1 are resistant to carbapenems and were predicted to become a major global health problem. To cope with this emerging threat, there is a need for rapid and sensitive molecular assays to detect bla _NDM-1 in carbapenem-resistant Enterobacteriaceae from clinical isolates. In diagnostic laboratories, real-time PCR is the current gold standard for the sensitive and rapid detection of pathogens. We describe a real-time PCR assay as well as two conventional PCR assays to detect bla _NDM-1. Only minute amounts of total DNA extracted from one bacterial colony are sufficient to allow detection of bla _NDM-1 by real-time PCR within less than 1 h. We also introduce a chemically synthesized bla _NDM-1 gene as a convenient positive control for those laboratories wishing to setup in-house assays for bla _NDM-1 detection. Importantly, our study represents a proof of principle for the usefulness of rapidly synthesized genes serving as positive controls for novel diagnostic PCR assays of emerging pathogens during the initial phase after their discovery when biological isolates are still rare and not commonly available.