School of Marine Science and Technology, Harbin Institute of Technology (Weihai), Weihai 264209, PR China; School of Environment, Harbin Institute of Technology, Harbin 150090, PR China.
School of Marine Science and Technology, Harbin Institute of Technology (Weihai), Weihai 264209, PR China; School of Marine Sciences, Ningbo University, Ningbo 315211, PR China.
Sci Total Environ. 2022 Jul 15;830:154771. doi: 10.1016/j.scitotenv.2022.154771. Epub 2022 Mar 24.
Prorocentrum minimum is a common diarrhetic shellfish toxins-producing marine microalga that may seriously endanger marine resources and cause great economic losses. The development of a novel rapid detection technique is of great importance for the prevention and control of the damage caused by P. minimum. In this study, the aptamer against P. minimum was for the first time generated from an artificially synthesized single-stranded DNA library by systematic evolution of ligand by exponential enrichment (SELEX), using P. minimum and P. minimum-related species, including Prorocentrum donghaiense, Prorocentrum lima and Prorocentrum micans as target and counter-screening species, respectively. The aptamer library was successfully obtained at the end of 18 rounds of SELEX-screening by continuously monitoring the binding ratio of the resultant ssDNA from each round. Three sequences (Apt 1, Apt 2 and Apt 3) with the highest frequency in the aptamer library resulted from high-throughput sequencing were first selected as candidate aptamers. The secondary structure of these sequences was predicted and analyzed. In addition, the specificity and affinity of these candidate aptamers were determined by flow cytometry analysis. The results indicated that these aptamers had high specificity and affinity, with a K of (224.6 ± 8.8) nM (Apt 1), (286.6 ± 13.9) nM (Apt 2) and (388.5 ± 44.6) nM (Apt 3), respectively. Apt 1 was therefore chosen as the best aptamer against P. minimum. Finally, the fluorescence microscopic examination further confirmed that Apt 1 can well bind to P. minimum. In summary, Apt 1 may be promising for being used as a novel molecular recognition element for P. minimum.
最小原甲藻是一种常见的产麻痹性贝类毒素的海洋微藻，可能严重危害海洋资源并造成巨大的经济损失。因此，开发一种新型快速检测技术对于预防和控制最小原甲藻造成的危害具有重要意义。本研究首次通过指数富集配体系统进化（SELEX）技术，从人工合成的单链 DNA 文库中筛选出针对最小原甲藻的适配体。以最小原甲藻及其相关种，包括东海原甲藻、利玛原甲藻和微小原甲藻，分别作为靶标和反筛物种，进行 SELEX 筛选。通过连续监测每轮产生的 ssDNA 的结合率，最终在 18 轮 SELEX 筛选后成功获得了适配体文库。从高通量测序得到的适配体文库中，首先选择出现频率最高的 3 个序列（Apt 1、Apt 2 和 Apt 3）作为候选适配体。预测和分析了这些序列的二级结构。此外，通过流式细胞术分析确定了这些候选适配体的特异性和亲和力。结果表明，这些适配体具有较高的特异性和亲和力，其 Kd 值分别为（224.6±8.8）nM（Apt 1）、（286.6±13.9）nM（Apt 2）和（388.5±44.6）nM（Apt 3）。因此，Apt 1 被选为针对最小原甲藻的最佳适配体。最后，荧光显微镜检查进一步证实 Apt 1 可以很好地与最小原甲藻结合。综上所述，Apt 1 有望成为最小原甲藻的新型分子识别元件。
