Multiplex Asymmetric PCR-Based Oligonucleotide Microarray for Detection of Drug Resistance Genes Containing Single Mutations in Enterobacteriaceae
Open Access
- 1 October 2007
- journal article
- research article
- Published by American Society for Microbiology in Antimicrobial Agents and Chemotherapy
- Vol. 51 (10), 3707-3713
- https://doi.org/10.1128/aac.01461-06
Abstract
A multiplex asymmetric PCR (MAPCR)-based microarray method was developed for the detection of 10 known extended-spectrum β-lactamases (ESBLs) and plasmid-mediated AmpC β-lactamase genes in gram-negative bacteria and for the typing of six important point mutations (amino acid positions 35, 43, 130, 179, 238, and 240) in the bla SHV gene. The MAPCR is based on a two-round reaction to promote the accumulation of the single-stranded amplicons amenable for microarray hybridization by employing multiple universal unrelated sequence-tagged primers and elevating the annealing temperature at the second round of amplification. A strategy to improve the discrimination efficiency of the microarray was constituted by introducing an artificial mismatch into some of the allele-specific oligonucleotide probes. The microarray assay correctly identified the resistance genes in both the reference strains and some 111 clinical isolates, and these results were also confirmed for some isolates by direct DNA sequence analysis. The resistance genotypes determined by the microarray correlated closely with phenotypic MIC susceptibility testing. This fast MAPCR-based microarray method should prove useful for undertaking important epidemiological studies concerning ESBLs and plasmid-mediated AmpC enzymes and could also prove invaluable as a preliminary screen to supplement phenotypic testing for clinical diagnostics.Keywords
This publication has 39 references indexed in Scilit:
- Regional variations in community consumption of antibiotics in Hungary, 1996–2003British Journal of Clinical Pharmacology, 2005
- Extended-Spectrum β-Lactamases: a Clinical UpdateClinical Microbiology Reviews, 2005
- The design and application of DNA chips for early detection of SARS-CoV from clinical samplesJournal of Clinical Virology, 2005
- An additional measure for quantifying antibiotic use in hospitalsJournal of Antimicrobial Chemotherapy, 2005
- Outpatient antibiotic use in Europe and association with resistance: a cross-national database studyThe Lancet, 2005
- Epidemiology and Clinical Features of Bloodstream Infections Caused by AmpC-Type-β-Lactamase-Producing Klebsiella pneumoniaeAntimicrobial Agents and Chemotherapy, 2004
- Sensitive Assay for Identification of Methicillin-Resistant Staphylococcus aureus, Based on Direct Detection of Genomic DNA by Use of Gold Nanoparticle ProbesClinical Chemistry, 2004
- Multiplex PCR combining deltaF508 mutation and intragenic microsatellites of the CFTR gene for pre-implantation genetic diagnosis (PGD) of cystic fibrosisEuropean Journal of Human Genetics, 2002
- Extended-Spectrum β-Lactamases in the 21st Century: Characterization, Epidemiology, and Detection of This Important Resistance ThreatClinical Microbiology Reviews, 2001
- Treatment options for extended-spectrum β-lactamase-producersFEMS Microbiology Letters, 2000