Biotechnology Department, Animal Health Research Institute, Agricultural Research Center, Dokki, Giza 12618, Egypt.
Institute of Bacterial Infections and Zoonoses, Friedrich-Loeffler-Institut, 07743 Jena, Germany.
Int J Mol Sci. 2022 May 30;23(11):6131. doi: 10.3390/ijms23116131.
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.
布鲁氏菌是革兰氏阴性、需氧、非运动的球杆菌，可引起人类和动物的布鲁氏菌病。该疾病是最严重但被忽视的全球动物源性传染病之一。特别是在发展中国家，布鲁氏菌病给私人动物所有者和国家收入带来了公共卫生问题和经济损失。适配子由寡核苷酸组成，是抗体的化学类似物，是开发基于适配子的快速、敏感和特异检测方法以鉴定该组细菌的有前途的成分。为此，通过指数富集的配体系统进化细胞（cell-SELEX）的增强方案生成和选择适配子。该增强的细胞-SELEX 程序结合了常规和 toggle 细胞-SELEX，以提高对整个细胞的特异性和结合亲和力。该程序与高通量测序相结合，对产生的适配子池进行全面的生物信息学分析和湿实验室验证实验，从而选择出一种对在埃及流行的那些种高度敏感和特异的适配子。分离出的候选适配子对 、 、 显示出解离常数（K）值分别为 43.5 ± 11、61.5 ± 8 和 56 ± 10.8 nM。据作者所知，这是首次使用常规和 toggle 细胞-SELEX 的增强组合开发针对 的特异性适配子。
