Microbiology & Fermentation Technology Department, CSIR-Central Food Technological Research Institute (CFTRI), Mysuru 570020, India.
Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh 201002, India.
J Agric Food Chem. 2022 May 25;70(20):6239-6246. doi: 10.1021/acs.jafc.2c01591. Epub 2022 May 12.
This study reports a novel bio-layer interferometry (BLI)-based SELEX for generation of high affinity aptamers against patulin. Unlike conventional SELEX, the present method enabled real-time monitoring of increasing affinity of the oligonucleotides to the toxin. After seven rounds of selection cycles, the enriched pool of aptamers was characterized by cloning and sequencing and clustered into two families based on similarity. Two sequences, PAT C3 and PAT C4, each belonging to different clades, were further evaluated for their binding affinity. SPR studies determined the dissociation constants () of 8.2 × 10 and 1.9 × 10 M for aptamer PAT C3 and PAT C4, respectively. The highest affinity PAT C3 aptamer was used to develop a patulin BLI aptasensor, which indicated a linear detection range from 0.045 to 100 ng/mL [limit of detection (LOD) = 0.173 ng/mL; limit of quantification (LOQ) = 0.526 ng/mL]. The aptasensor displayed no cross-reactivity with its structural analogue isopatulin or any of the other mycotoxin groups tested. Spiking studies in simulated apple juice samples showed recoveries in the range of 82.11 to 100.23%, indicating good sensor performance. The study is the first report of BLI-based SELEX for a non-protein toxin, which resulted in the generation of high affinity aptamers and development of an aptasensor which can have wide application in the food industry for high throughput screening of samples for patulin contamination within a short span of time.
本研究报告了一种基于生物层干涉（BLI）的新型 SELEX 方法，用于生成针对棒曲霉素的高亲和力适体。与传统 SELEX 不同，本方法能够实时监测寡核苷酸与毒素结合亲和力的增加。经过七轮选择循环，富集的适体库通过克隆和测序进行表征，并根据相似性聚类为两个家族。根据序列相似性，将两条序列，PAT C3 和 PAT C4，分别聚类到不同的分支中。进一步评估了这两个序列的结合亲和力。SPR 研究确定适体 PAT C3 和 PAT C4 的解离常数（Kd）分别为 8.2×10-8 和 1.9×10-7 M。最高亲和力的 PAT C3 适体被用于开发棒曲霉素 BLI 适体传感器，其检测范围线性为 0.045 至 100 ng/mL [检测限（LOD）= 0.173 ng/mL；定量限（LOQ）= 0.526 ng/mL]。该适体传感器与结构类似物异棒曲霉素或测试的任何其他霉菌毒素组均无交叉反应性。在模拟苹果汁样品中的加标研究表明回收率在 82.11%至 100.23%之间，表明传感器性能良好。本研究是首例基于 BLI 的非蛋白毒素 SELEX 的报道，该方法产生了高亲和力的适体，并开发了一种适体传感器，该传感器可广泛应用于食品工业，用于在短时间内高通量筛选样品中的棒曲霉素污染。
