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

被引:11
作者
El-Husseini, Dalia M. [1 ,2 ]
Sayour, Ashraf E. [3 ]
Melzer, Falk [2 ]
Mohamed, Magda F. [4 ]
Neubauer, Heinrich [2 ]
Tammam, Reham H. [4 ]
机构
[1] Agr Res Ctr, Anim Hlth Res Inst, Biotechnol Dept, Giza 12618, Egypt
[2] Friedrich Loeffler Inst, Inst Bacterial Infect & Zoonoses, D-07743 Jena, Germany
[3] Agr Res Ctr, Anim Hlth Res Inst, Mol Biomimet Res Grp, Giza 12618, Egypt
[4] Cairo Univ, Fac Sci, Chem Dept, Giza 12613, Egypt
关键词
Brucella; aptamer; enhanced cell-SELEX; qPCR; high-throughput sequencing; SINGLE-STRANDED-DNA; MOLECULAR PROBES; IN-VITRO; IDENTIFICATION; PROGRESS; DESIGN; OPTIMIZATION; STRATEGY; BINDING;
D O I
10.3390/ijms23116131
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 Brucella 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 Brucella 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 Brucella species known to circulate in Egypt. The isolated candidate aptamer showed dissociation constant (K-D) values of 43.5 +/- 11, 61.5 +/- 8, and 56 +/- 10.8 nM for B. melitensis, B. abortus, and B. suis, respectively. This is the first development of a Brucella-specific aptamer using an enhanced combination of conventional and toggle cell-SELEX to the authors' best knowledge.
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页数:20
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