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Selection of DNA aptamers against uropathogenic Escherichia coli NSM59 by quantitative PCR controlled Cell-SELEX
- Source :
- Journal of Microbiological Methods. 104:94-100
- Publication Year :
- 2014
- Publisher :
- Elsevier BV, 2014.
-
Abstract
- In order to better control nosocomial infections, and facilitate the most prudent and effective use of antibiotics, improved strategies for the rapid detection and identification of problematic bacterial pathogens are required. DNA aptamers have much potential in the development of diagnostic assays and biosensors to address this important healthcare need, but further development of aptamers targeting common pathogens, and the strategies used to obtain specific aptamers are required. Here we demonstrate the application of a quantitative PCR (qPCR) controlled Cell-SELEX process, coupled with downstream secondary-conformation-based aptamer profiling. We used this approach to identify and select DNA aptamers targeted against uropathogenic Escherichia coli, for which specific aptamers are currently lacking, despite the prevalence of these infections. The use of qPCR to monitor the Cell-SELEX process permitted a minimal number of SELEX cycles to be employed, as well as the cycle-by-cycle optimisation of standard PCR amplification of recovered aptamer pools at each round. Identification of useful aptamer candidates was also facilitated by profiling of secondary conformations and selection based on putative aptamer secondary structure. One aptamer selected this way (designated EcA5-27), displaying a guanine-quadruplex sequence motif, was shown to have high affinity and specificity for target cells, and the potential to discriminate between distinct strains of E. coli, highlighting the possibility for development of aptamers selectively recognising pathogenic strains. Overall, the identified aptamers hold much potential for the development of rapid diagnostic assays for nosocomial urinary tract infections caused by E. coli.
- Subjects :
- Microbiology (medical)
Aptamer
Molecular Sequence Data
Computational biology
Cell selex
Biology
DNA Aptamers
medicine.disease_cause
Polymerase Chain Reaction
Microbiology
law.invention
law
medicine
Humans
Uropathogenic Escherichia coli
Molecular Biology
Escherichia coli
Escherichia coli Infections
Polymerase chain reaction
Base Sequence
SELEX Aptamer Technique
Aptamers, Nucleotide
Virology
Real-time polymerase chain reaction
Nucleic Acid Conformation
Sequence motif
Systematic evolution of ligands by exponential enrichment
Subjects
Details
- ISSN :
- 01677012
- Volume :
- 104
- Database :
- OpenAIRE
- Journal :
- Journal of Microbiological Methods
- Accession number :
- edsair.doi.dedup.....c1c3b081df496f25e6c7a16e5a8d325a