Selection and identification of DNA aptamers targeted to Vibrio parahemolyticus based on whole-bacterium SELEX

  Objective  To obtain the DNA aptamers specific binding to Vibrio parahemolyticus (VP) for rapid and sensitive detection of VP.

  Methods  The oligonucleotide library was synthesized as a single-stranded 86-mer, with 40-mer random oligonucleotides in center for whole-bacterium systematic evolution of ligands by exponential enrichment (SELEX) targeted to VP. Determination of specificity and affinity of the library were then carried out for the candidate aptamers. Prediction of secondary structures and estimation of binding dissociation constants (Kd) of the aptamers were also performed.

  Results  The aptamer pools from the twelfth round of SELEX displayed the highest affinity for the target cells. The aptamer pools were cloned and sequenced, and a total of 5 sequences were obtained as the candidate aptamers (F2, F5, F6, F19, and F30); the binding affinity of these sequences were 68.8%, 68.8%, 73.1%, 65.2%, and 72.7%. All of these aptamer sequences showed preferential binding to VP over the other 5 types of bacteria. Predictions of secondary structures with DNA folding form demonstrated that all the five candidate aptamers were with several stem rings based on G-C base pairing. Kd value of these aptamers' sequences were 15.88, 15.24, 9.13, 4.12, and 25.39, respectively.

  Conclusion  The use of whole-bacterium SELEX to identify DNA aptamers that are specific for Vibrio parahemolyticus is feasible. All the 5 selected candidate aptamers are of high affinity and specificity, and are suitable for further development of rapid detection.