title: Rapid detection of TEM-type extended-spectrum β-lactamase (ESBL) mutations using lights-on/lights-off probes with single-stranded DNA amplification.
abstract: Rapid identification of specific TEM-type β-lactamase genes in bacterial infections is important for determining appropriate clinical treatment. We report here the design and initial testing of a molecular diagnostic assay capable of amplifying a large segment of the blaTEM gene, as well as detecting widely spaced extended-spectrum β-lactamase (ESBL) mutations and inhibitor-resistant TEM (IRT) mutations (eg, clavulanic acid resistance). Single-stranded DNA is generated using linear-after-the-exponential PCR (LATE-PCR) and is analyzed at the endpoint, using a set of four fluorescently labeled and four quencher-labeled probes in a single closed tube. These lights-on/lights-off probes work in concert to generate sequence-specific fluorescence contours over a temperature range from 25°C to 75°C. Mutant sequences from synthetic TEM gene variants and from TEM gene variants in bacterial strains generated large increases in fluorescent signal relative to that from the reference sequence for TEM-1. Clinical use of this convenient, single-closed-tube assay would make it possible to rapidly distinguish ESBL from non-ESBL variants and thereby to begin early treatment with suitable antibiotics.
authors.affiliation: Department of Biology, Brandeis University, Waltham, MA 02454, USA. pierce@brandeis.edu
authors.forename: Kenneth E
authors.initials: KE
authors.lastname: Pierce
authors.forename: Harald
authors.initials: H
authors.lastname: Peter
authors.forename: Till T
authors.initials: TT
authors.lastname: Bachmann
authors.forename: Carmelo
authors.initials: C
authors.lastname: Volpe
authors.forename: Rohit
authors.initials: R
authors.lastname: Mistry
authors.forename: John E
authors.initials: JE
authors.lastname: Rice
authors.forename: Lawrence J
authors.initials: LJ
authors.lastname: Wangh
mesh_terms: D017931:DNA Primers; D015342:DNA Probes / Q000378:metabolism; D004269:DNA, Bacterial / Q000302:isolation & purification*; D004277:DNA, Single-Stranded; D004755:Enterobacteriaceae / Q000201:enzymology / Q000302:isolation & purification; D005456:Fluorescent Dyes; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D001618:beta-Lactamases / Q000235:genetics* / Q000302:isolation & purification*
chemical_list: D017931:DNA Primers; D015342:DNA Probes; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D005456:Fluorescent Dyes; D001618:beta-Lactamases