This study describes ongoing efforts to identify and isolate specific DNA aptamers that bind to p57 also known as Major Soluble Antigen (MSA), the principal antigenic protein found on the cell surface of the Rs bacterium, responsible for causing bacterial kidney disease (BKD) in salmonids. Two different templates, a 80-mer with a 40 bp random region and a 100-mer with a 60 bp random region, were used. Six sub constructs were constructed from the full length MSA protein based on three selected regions. Aptamers were identified from a very large, random pool through a process called Systematic Evolution of Ligands by EXponential amplification (SELEX). SELEX was performed on the R2c sub construct from region 2 which contains a putative DNA-binding domain and therefore is believed to be a good binding site for aptamers. Prior to each round of SELEX, ssDNA was isolated by reacting the biotin-labeled dsDNA with streptavidin coated sepharose beads. The separation is made possible by the fact that biotin binds strongly to streptavidin. The ssDNA was recovered by denaturing the biotinylated dsDNA with sodium hydroxide. Once isolated and characterized by DNA sequencing, aptamers will be used in binding assays to identify those with the highest affinity to R2c binding site with the purpose of developing a biosensor for Rs detection in water.

Selection of protein-binding DNA aptamers for bacterial detection

249th National Meeting (2015)

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