Rapid Detection of TEM-Type Extended-Spectrum B-Lactamase (ESBL) Mutations Using Lights-On/Lights-Off Probes with Single-Stranded DNA Amplification

• Autores: Kenneth E. Pierce, Harald Peter, Till T. Bachmann, Carmelo Volpe, Rohit Mistry, John E. Rice, Lawrence J. Wangh
• Localización: The Journal of molecular diagnostics, ISSN 1525-1578, Vol. 15, Nº. 3, 2013, págs. 291-298
• Idioma: inglés
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• Resumen

  • Rapid identification of specific TEM-type B-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 B-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.