Multiplex real-time PCR assay for detection and classification of Klebsiella pneumoniae carbapenemase gene (bla_KPC) variants

Carbapenem resistance mediated by plasmid-borne Klebsiella pneumoniae carbapenemases (KPC) is an emerging problem of significant clinical importance in Gram-negative bacteria. Multiple KPC gene variants (bla_KPC) have been reported, with KPC-2 (bla_KPC-2) and KPC-3 (bla_KPC-3) associated with epidemic outbreaks in New York City and various international settings. Here, we describe the development of a multiplex real-time PCR assay using molecular beacons (MB-PCR) for rapid and accurate identification of bla_KPC variants. The assay consists of six molecular beacons and two oligonucleotide primer pairs, allowing for detection and classification of all currently described bla_KPC variants (bla_KPC-2 to bla _KPC-11). The MB-PCR detection limit was 5 to 40 DNA copies per reaction and 4 CFU per reaction using laboratory-prepared samples. The MB-PCR probes were highly specific for each bla_KPC variant, and cross-reactivity was not observed using DNA isolated from several bacterial species. A total of 457 clinical Gram-negative isolates were successfully characterized by our MB-PCR assay, with bla_KPC-3 and bla _KPC-2 identified as the most common types in the New York/New Jersey metropolitan region. The MB-PCR assay described herein is rapid, sensitive, and specific and should be useful for understanding the ongoing evolution of carbapenem resistance in Gram-negative bacteria. As novel bla_KPC variants continue to emerge, the MB-PCR assay can be modified in response to epidemiologic developments.