Hubei Key Laboratory of Edible Wild Plants Conservation and Utilization, Hubei Normal University, Cihu Road, Huangshigang District, Huangshi 435002, China.
Guangdong Provincial Key Laboratory of Food Quality and Safety, South China Agricultural University, Wushan Road, Tianhe District, Guangzhou 510642, China.
Molecules. 2018 Sep 13;23(9):2337. doi: 10.3390/molecules23092337.
We describe a multiple combined strategy to discover novel aptamers specific for clenbuterol (CBL). An immobilized ssDNA library was used for the selection of specific aptamers using the systematic evolution of ligands by exponential enrichment (SELEX). Progress was monitored using real-time quantitative PCR (Q-PCR), and the enriched library was sequenced by high-throughput sequencing. Candidate aptamers were picked and preliminarily identified using a gold nanoparticles (AuNPs) biosensor. Bioactive aptamers were characterized for affinity, circular dichroism (CD), specificity and sensitivity. The Q-PCR amplification curve increased and the retention rate was about 1% at the eighth round. Use of the AuNPs biosensor and CD analyses determined that six aptamers had binding activity. Affinity analysis showed that aptamer 47 had the highest affinity (Kd = 42.17 ± 8.98 nM) with no cross reactivity to CBL analogs. Indirect competitive enzyme linked aptamer assay (IC-ELAA) based on a 5'-biotin aptamer 47 indicated the limit of detection (LOD) was 0.18 ± 0.02 ng/L (n = 3), and it was used to detect pork samples with a mean recovery of 83.33⁻97.03%. This is the first report of a universal strategy including library fixation, Q-PCR monitoring, high-throughput sequencing, and AuNPs biosensor identification to select aptamers specific for small molecules.
我们描述了一种多重联合策略，用于发现针对克仑特罗（CBL）的新型适体。使用固定化的 ssDNA 文库，通过指数富集的配体系统进化（SELEX）进行特定适体的选择。使用实时定量 PCR（Q-PCR）监测进展情况，并通过高通量测序对富集文库进行测序。使用金纳米粒子（AuNPs）生物传感器挑选和初步鉴定候选适体。使用 AuNPs 生物传感器和 CD 分析对生物活性适体进行亲和力、圆二色性（CD）、特异性和灵敏度的表征。在第八轮时，Q-PCR 扩增曲线增加，保留率约为 1%。使用 AuNPs 生物传感器和 CD 分析确定有六个适体具有结合活性。亲和力分析表明，适体 47 具有最高的亲和力（Kd = 42.17 ± 8.98 nM），与 CBL 类似物无交叉反应。基于 5'-生物素适体 47 的间接竞争酶联适体测定法（IC-ELAA）表明，检测限（LOD）为 0.18 ± 0.02 ng/L（n = 3），并用于检测猪肉样品，平均回收率为 83.33⁻97.03%。这是首次报道包括文库固定化、Q-PCR 监测、高通量测序和 AuNPs 生物传感器鉴定在内的用于选择针对小分子的特异性适体的通用策略。
