1 Multiplex Real-Time PCR Assay for Detection and Classification of Klebsiella 1 pneumoniae Carbapenemase Gene (blaKPC) Variants 2 3

2 Abstract 28 29 Carbapenem resistance mediated by plasmid-borne Klebsiella pneumoniae carbapenemases 30 (KPC) is an emerging problem of significant clinical importance in Gram-negative bacteria. 31 Multiple KPC gene variants (blaKPC) have been reported, with KPC-2 (blaKPC-2) and KPC-3 32 (blaKPC-3) associated with epidemic outbreaks in New York City and various international 33 settings. Herein we describe the development of a multiplex real-time PCR assay using 34 molecular beacons (MB-PCR) for rapid and accurate identification of blaKPC variants. The 35 assay consists of six molecular beacons and two oligonucleotide primer pairs, allowing for 36 detection and classification of all currently described blaKPC variants (blaKPC-2 to blaKPC-11). 37 The MB-PCR detection limit was 5-40 DNA copies per reaction, and 4 colony-forming units 38 (CFU) per reaction using laboratory-prepared samples. The MB-PCR probes were highly 39 specific for each blaKPC variant, and cross reactivity was not observed using DNA isolated 4