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.
Public Health Research Institute Tuberculosis Center, International Center for Public Health, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103, USA.
Liang
L
Chen
José R
JR
Mediavilla
Andrea
A
Endimiani
Marnie E
ME
Rosenthal
Yanan
Y
Zhao
Robert A
RA
Bonomo
Barry N
BN
Kreiswirth
D001426:Bacterial Proteins / Q000145:classification* / Q000235:genetics*; D001431:Bacteriological Techniques / Q000379:methods*; D003429:Cross Reactions; D017931:DNA Primers / Q000235:genetics; D006090:Gram-Negative Bacteria / Q000201:enzymology* / Q000235:genetics; D016905:Gram-Negative Bacterial Infections / Q000382:microbiology*; D006801:Humans; D009515:New Jersey; D009519:New York City; D015345:Oligonucleotide Probes / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D001618:beta-Lactamases / Q000145:classification* / Q000235:genetics*
D001426:Bacterial Proteins; D017931:DNA Primers; D015345:Oligonucleotide Probes; D001618:beta-Lactamases; C063912:carbapenemase