ISOLATION AND CHARACTERIZATION OF HIGH AFFINITY DNA APTAMERS FOR DETECTION OF VIBRIO CHOLERAE

Background and Aim:In recent years, the prevalence diseases caused by cholera spp is increasing in the world and among these species, Vibrio cholerae is the most important Vibrio associated with pandemic and epidemic cholera outbreaks. Therefore, the development of a reliable method for early and accurate detection of V.cholerae for management of diseases is a real need. Aptamers with the ability to detect targets with high specificity and accuracy can be one of the candidates used for whole cell and there by V. Cholerae detection.Methods:In this research high affinity DNA aptamers against V. cholerae O1 with two major serotypes of Inaba and Ogawa were selected from DNA aptamer library through 12 rounds of Systematic Evolution of Ligands by Exponential (SELEX) enrichment procedure using live cells as a target. The aptamer pool of 12th round was cloned and transferred into the E. coli DH5α cells. A total of 67 aptamer transformants were attained and were approved as positive clones with colony PCR. Thirteen positive clones were analyzed by flow cytometry and three clones with the highest fluorescent intensity were chosen for additional analysis.Results:During the SELEX rounds, the binding efficiency showed a significant growth from the second round (4.44%) to the twelfth round (50.9%). Our results showed descending affinity toward counter cells.Conclusion:In conclusion, the isolated aptamers can be used in any tool like biosensor for detection of V. cholerae.

Keywords:

Aptamer , V. cholerae O1 , Cell-SELEX , Flow Cytometry