Selection of molecular aptamers for identification of live cells of Ralstonia solanacearum: A new method in plant pathology.
P. G. CHAMPOISEAU (1), J. B. Jones (1), K. Sefah (2), W. Tan (2) (1) University of Florida, Department of Plant Pathology, Gainesville, FL, USA; (2) University of Florida, Department of Chemistry, Gainesville, FL, USA.
Phytopathology 99:S20.

Abstract: Ralstonia solanacearum (Rs) has the potential to be one of the most damaging plant pathogenic bacteria in production fields of diverse crops. One subgroup of Rs, designated race 3 biovar 2 (R3bv2), is on the select agent list and is subject to the strictest biosecurity regulations in the United States. Due to its status, the need for unambiguous identification of R3bv2 is critical; however, diagnostic tools currently used by official laboratories lack the requisite sensitivity, specificity, or speed. Molecular aptamers are single-stranded oligonucleotides that can specifically bind with high affinity to a variety of molecules ranging from macromolecules to small compounds. They are generated through an in vitro selection process termed SELEX (system evolution of ligands by exponential enrichment) and have been used quasi-exclusively in human cancer research. Recently, a cell-SELEX protocol was developed to successfully produce aptamers that could specifically differentiate two closely-related cell lines using whole live cells. In this study, we used Rs race 3 strain UW485 (target cells) and race 1 strain Rs5 (control cells) to evaluate the potential for cell-SELEX to produce Rs race 3-specific aptamers. After 16 rounds of selection, flow-cytometry data indicated enrichment of aptamers specific to the UW485 strain, with evidence for targeting cell exopolysaccharides. Aptamers potentially could be produced and used in the future as reproducible, fast and highly-specific diagnostic tools for R3bv2.