title: Real-time TaqMan PCR for rapid detection of genes encoding five types of non-metallo- (class A and D) carbapenemases in Enterobacteriaceae.
abstract: A real-time TaqMan multiplex polymerase chain reaction (PCR) assay was developed to detect genes encoding five types of serine carbapenemases (GES, IMI/NMC, KPC, OXA-48 and SME). The assay was validated using control strains known to produce each of these types of enzyme and was then further assessed by 'blindly' testing 59 previously characterised clinical isolates, including 19 with serine (KPC or OXA-48) carbapenemases, 22 with metallo- (IMP, VIM or NDM) carbapenemases, and 18 with carbapenem resistance contingent upon extended-spectrum β-lactamase (ESBL) or AmpC production combined with porin loss. The assay detected and correctly assigned the serine carbapenemases in all five positive control strains and in 19 clinical isolates. No false-positive results were seen for isolates with metallo-enzymes or for those that lacked a carbapenemase. The five serine carbapenemase genotypes could also be distinguished by melt-curve analysis or the molecular size of the amplicons.
authors.affiliation: Clinical Microbiology and Public Health Laboratory, Addenbrooke's Hospital, Cambridge CB2 0QW, UK. rosemary.swayne@addenbrookes.nhs.uk
authors.forename: R L
authors.initials: RL
authors.lastname: Swayne
authors.forename: H A
authors.initials: HA
authors.lastname: Ludlam
authors.forename: V G
authors.initials: VG
authors.lastname: Shet
authors.forename: N
authors.initials: N
authors.lastname: Woodford
authors.forename: M D
authors.initials: MD
authors.lastname: Curran
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics*; D015780:Carbapenems / Q000494:pharmacology; D002850:Chromatography, Gel; D017931:DNA Primers; D004755:Enterobacteriaceae / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics* / Q000302:isolation & purification; D004756:Enterobacteriaceae Infections / Q000382:microbiology; D006801:Humans; D008826:Microbial Sensitivity Tests; D016133:Polymerase Chain Reaction / Q000379:methods*; D019914:Taq Polymerase; D013997:Time Factors; D044366:Transition Temperature; D018440:beta-Lactam Resistance / Q000235:genetics*; D001618:beta-Lactamases / Q000235:genetics*; D047090:beta-Lactams / Q000494:pharmacology
chemical_list: D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D015780:Carbapenems; D017931:DNA Primers; D047090:beta-Lactams; D019914:Taq Polymerase; D001618:beta-Lactamases; C063912:carbapenemase