Duan Ye, Gao Zhiqiang, Wang Lihui, Wang Huishan, Zhang Hexiao, Li Hao
Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, China.
Beijing Entry-Exit Inspection and Quarantine Bureau, Beijing, 100026, China.
Appl Biochem Biotechnol. 2016 Dec;180(8):1644-1656. doi: 10.1007/s12010-016-2193-6. Epub 2016 Sep 9.
Chloramphenicol (CAP) has been widely used to treat bacterial infections in livestock and aquatic animals. To reduce the risk of CAP residues, an efficient technology to rapidly detect CAP residues in animal-sourced food is expressly needed. In this study, magnetic bead-based systematic evolution of ligands by exponential enrichment (Mag-SELEX) strategy was performed to select and identify CAP-specific single-stranded DNA (ssDNA) aptamers from a random oligonucleotide library. After nine rounds of selection, five potential ssDNA aptamers were selected. Low homology indicated that they might belong to different families. To identify an aptamer with the highest affinity for CAP, the dissociation constant (K ) values of these selected aptamers were determined. The lowest K values of two potential aptamers (i.e., No. 4 and No. 5) were, respectively, 0.10162 ± 0.0111 and 0.03224 ± 0.00819 μM, which were much lower than previously reported lowest K value (i.e., 0.766 μM) of CAP aptamer. Moreover, compared with No. 4, aptamer No. 5 had higher binding rate, which is quite different among those with CAP and with CAP's structural analogs (i.e., thiamphenicol (TAP) and florfenicol (FF)). These results indicated that the potential aptamer No. 5 with highest specificity and affinity for CAP would be an ideal aptamer for future detection of residual CAP in animal-sourced food.
氯霉素（CAP）已被广泛用于治疗家畜和水生动物的细菌感染。为降低CAP残留风险，迫切需要一种能快速检测动物源食品中CAP残留的高效技术。在本研究中，采用基于磁珠的指数富集配体系统进化（Mag-SELEX）策略，从随机寡核苷酸文库中筛选并鉴定CAP特异性单链DNA（ssDNA）适配体。经过九轮筛选，选出了五个潜在的ssDNA适配体。低同源性表明它们可能属于不同家族。为鉴定对CAP亲和力最高的适配体，测定了这些筛选出的适配体的解离常数（K）值。两个潜在适配体（即4号和5号）的最低K值分别为0.10162±0.0111和0.03224±0.00819μM，远低于先前报道的CAP适配体最低K值（即0.766μM）。此外，与4号相比，5号适配体具有更高的结合率，这在与CAP及其结构类似物（即甲砜霉素（TAP）和氟苯尼考（FF））结合时差异显著。这些结果表明，对CAP具有最高特异性和亲和力的潜在5号适配体将是未来检测动物源食品中CAP残留的理想适配体。
