Generation and Selection of Specific Aptamers Targeting Species through an Enhanced Cell-SELEX Methodology.

Brucellae are Gram-negative, aerobic, non-motile coccobacilli causing brucellosis in man and animals. The disease is one of the most significant yet neglected global zoonoses. Especially in developing countries, brucellosis is causing public health problems and economic losses to private animal owners and national revenues. Composed of oligonucleotides, aptamers are chemical analogues of antibodies that are promising components for developing aptamer-based rapid, sensitive, and specific tests to identify the group of bacteria. For this purpose, aptamers were generated and selected by an enhanced protocol of cell systematic evolution of ligands by exponential enrichment (cell-SELEX). This enhanced cell-SELEX procedure involved the combination of both conventional and toggle cell-SELEX to boost the specificity and binding affinity to whole cells. This procedure, combined with high-throughput sequencing of the resulting aptamer pools, comprehensive bioinformatics analysis, and wet lab validation assays, led to the selection of a highly sensitive and specific aptamer for those species known to circulate in Egypt. The isolated candidate aptamer showed dissociation constant (K) values of 43.5 ± 11, 61.5 ± 8, and 56 ± 10.8 nM for , , and , respectively. This is the first development of a -specific aptamer using an enhanced combination of conventional and toggle cell-SELEX to the authors' best knowledge.