Selection and identification of a DNA aptamer targeted to Vibrio parahemolyticus.

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 . FAM-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.

[1]  J. Szostak,et al.  In vitro selection of RNA aptamers specific for cyanocobalamin. , 1994, Biochemistry.

[2]  C. R. Osorio,et al.  Identification of tdh-positive Vibrio parahaemolyticus from an outbreak associated with raw oyster consumption in Spain. , 2003, FEMS microbiology letters.

[3]  B. Shen,et al.  Combining use of a panel of ssDNA aptamers in the detection of Staphylococcus aureus , 2009, Nucleic acids research.

[4]  H. Oikawa,et al.  Occurrence and density of vibrio parahaemolyticus in live edible crustaceans from markets in China. , 2006, Journal of food protection.

[5]  Michael Famulok,et al.  Functional aptamers and aptazymes in biotechnology, diagnostics, and therapy. , 2007, Chemical reviews.

[6]  X Chris Le,et al.  Selection of aptamers against live bacterial cells. , 2008, Analytical chemistry.

[7]  J. Wells,et al.  Seasonal Abundance of Total and Pathogenic Vibrio parahaemolyticus in Alabama Oysters , 2003, Applied and Environmental Microbiology.

[8]  J. Wrzesinski,et al.  Characterization of structure and metal ions specificity of Co2+-binding RNA aptamers. , 2005, Biochemistry.

[9]  F. Luo,et al.  Aptamer from whole-bacterium SELEX as new therapeutic reagent against virulent Mycobacterium tuberculosis. , 2007, Biochemical and biophysical research communications.

[10]  M. Famulok,et al.  RNA aptamers specifically interact with the prion protein PrP , 1997, Journal of virology.

[11]  S. Rippey,et al.  Infectious diseases associated with molluscan shellfish consumption , 1994, Clinical Microbiology Reviews.

[12]  Kathryn J Boor,et al.  Epidemiology, pathogenesis, and prevention of foodborne Vibrio parahaemolyticus infections. , 2004, Foodborne pathogens and disease.

[13]  E. Vermaas,et al.  Selection of single-stranded DNA molecules that bind and inhibit human thrombin , 1992, Nature.

[14]  X. Le,et al.  DNA aptamers binding to multiple prevalent M-types of Streptococcus pyogenes. , 2011, Analytical chemistry.

[15]  L. Gold,et al.  Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. , 1990, Science.

[16]  A D Ellington,et al.  Aptamers as therapeutic and diagnostic reagents: problems and prospects. , 1997, Current opinion in chemical biology.

[17]  M. Famulok,et al.  Oligonucleotide aptamers that recognize small molecules. , 1999, Current opinion in structural biology.

[18]  J. Szostak,et al.  In vitro selection of RNA molecules that bind specific ligands , 1990, Nature.