Rapid Fluorescent Detection of Escherichia coli K88 Based on DNA Aptamer Library as Direct and Specific Reporter Combined With Immuno-Magnetic Separation
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Nucleic acid aptamers have long demonstrated the capacity to bind cells with high affinity so that they have been utilized to diagnose various important pathogens. In this study, a DNA aptamer library was on initial efforts developed to act as a specific reporter for rapid detection of enter toxigenic Escherichia coli (ETEC) K88 combined with immuno-magnetic separation (IMS). During a Whole-cell Systematic Evolution of Ligands by Exponential Enrichment (CELL-SELEX) procedure, the last selection pool against ETEC K88, which is named “DNA aptamer library” here, was selected and subsequently identified by flow cytometric analysis and confocal imaging. A K88 monoclonal antibody (mAb) with high affinity (Kaff: 1.616 ± 0.033 × 108 M−1) against K88 fimbrial protein was prepared, biotinylated and conjugated to streptavidin-coated magnetic beads (MBs). After the bacteria were effectively captured and enriched from the complex sample by immuno-magnetic beads (IMBs), 5′-FITC modified aptamer library was directly bound to target cells as a specific reporter for its detection. The detection system showed clearly high specificity and sensitivity with the detection limit of 1.1 × 103 CFU/ml in pure culture and 2.2 × 103 CFU/g in artificially contaminated fecal sample. The results also indicated that fluorophore-lablled DNA aptamer library as specific reporter could generate more reliable signals than individual aptamer with best affinity against target cells and implied it would have great applied potential in directly reporting bacteria from complex samples combined with IMS technology.
KeywordsETEC K88 detection IMS Aptamer library Reporter
We would like to thank the National Natural Science Foundation (81,271,660), Ministry of Education for the Doctoral Program of Higher Education (20,114,306,110,006), and Scientific Research Fund of Hunan Provincial Education Department (09 K021, 12 K032) for financial support.
Conflict of Interest
The authors declare that there are no conflicts of interest, financial or otherwise, associated with the publication of this study.
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