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Establishment of a Simple and Quick Method for Detecting Extended-Spectrum β-Lactamase (ESBL) Genes in Bacteria
- Source :
- Journal of Biomolecular Techniques : JBT. 27:132-137
- Publication Year :
- 2016
- Publisher :
- Association of Biomolecular Resource Facilities, 2016.
-
Abstract
- Extended-spectrum β-lactamase (ESBL) genes that render bacteria resistant to antibiotics are commonly detected using phenotype testing, which is time consuming and not sufficiently accurate. To establish a better method, we used phenotype testing to identify ESBL-positive bacterial strains and conducted PCR to screen for TEM (named after the patient Temoneira who provided the first sample), sulfhydryl reagent variable (SHV), cefotaxime (CTX)-M-1, and CTX-M-9, the 4 most common ESBL types and subtypes. We then performed multiplex PCR with 1 primer containing a biotin and hybridized the PCR products with gene-specific probes that were coupled with microbeads and coated with a specific fluorescence. The hybrids were linked to streptavidin-R-phycoerythrins (SA-PEs) and run through a flow cytometer, which sorted the fluorescently dyed microbeads and quantified the PEs. The results from single PCR, multiplex PCR, and cytometry were consistent with each other. We used this method to test 169 clinical specimens that had been determined for phenotypes and found 154 positive for genotypes, including 30 of the 45 samples that were negative for phenotypes. The CTX-M genotype tests alone, counting both positive and negative cases, showed 99.41% (168/169) consistency with the ESBL phenotype test. Thus, we have established a multiplex-PCR system as a simple and quick method that is high throughput and accurate for detecting 4 common ESBL types and subtypes.
- Subjects :
- Cefotaxime
0208 environmental biotechnology
02 engineering and technology
010501 environmental sciences
Biology
01 natural sciences
Article
beta-Lactam Resistance
beta-Lactamases
Microbiology
Genotype
Sulfhydryl reagent
Multiplex polymerase chain reaction
Escherichia coli
medicine
Humans
Molecular Biology
Escherichia coli Infections
0105 earth and related environmental sciences
Escherichia coli Proteins
Reproducibility of Results
biology.organism_classification
Phenotype
Molecular biology
020801 environmental engineering
Klebsiella pneumoniae
Molecular Diagnostic Techniques
Genes, Bacterial
Primer (molecular biology)
Multiplex Polymerase Chain Reaction
Cytometry
Bacteria
medicine.drug
Subjects
Details
- ISSN :
- 19434731 and 15240215
- Volume :
- 27
- Database :
- OpenAIRE
- Journal :
- Journal of Biomolecular Techniques : JBT
- Accession number :
- edsair.doi.dedup.....b21516457b8006cbaae5dfc7b086179b