Establishment of a Simple and Quick Method for Detecting Extended-Spectrum β-Lactamase (ESBL) Genes in Bacteria
| Autor: | Liao Dj, Fei Y, Chen Lc, Xu M, Han St, Huang Jy, Tan Yj |
|---|---|
| Rok vydání: | 2016 |
| Předmět: |
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 |
| Zdroj: | Journal of Biomolecular Techniques : JBT. 27:132-137 |
| ISSN: | 1943-4731 1524-0215 |
| DOI: | 10.7171/jbt.16-2704-001 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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