期刊
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
卷 60, 期 16, 页码 4034-4038出版社
AMER CHEMICAL SOC
DOI: 10.1021/jf300395z
关键词
aptamer; Vibrio parahemolyticus; SELEX; flow cytometry
资金
- Science and Technology Supporting Project of Jiangsu Province [BE2011621, BE2010679]
- National S&T support program of China [2012BAK08B01]
- Merieux Research Grant Program
- Research Fund for the Doctoral Program of Higher Education [20110093110002]
- Program for New Century Excellent Talents in University [NCET-11-0663]
A whole-bacterium systemic evolution of ligands by exponential enrichment (SELEX) method was applied to a combinatorial library of FAM-labeled single-stranded DNA molecules to identify DNA aptamers demonstrating specific binding to Vibrio parahemolyticus. PAM-labeled aptamer sequences with high binding affinity to V. parahemolyticus were identified by flow cytometric analysis. Aptamer A3P, which showed a particularly high binding affinity in preliminary studies, was chosen for further characterization. This aptamer displayed a dissociation constant (K-d) of 16.88 +/- 1.92 nM. Binding assays to assess the specificity of aptamer A3P showed a high binding affinity (76%) for V. parahemolyticus and a low apparent binding affinity (4%) for other bacteria. Whole-bacterium SELEX is a promising technique for the design of aptamer-based molecular probes for microbial pathogens that does not require the labor-intensive steps of isolating and purifying complex markers or targets.
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