甲胺磷的核酸适体筛选

Selection of aptamer against methamidophos

中文关键词:  甲胺磷  适体  食品安全  检测技术

英文关键词:methamidophos  aptamer  food safety  detection technology

中文摘要:

      目的 筛选识别甲胺磷的DNA适体。方法 体外合成ssDNA 文库, 将其固相化于琼脂糖凝胶颗粒表面, 液相中甲胺磷与其结合的DNA分子从固相表面分离, 进而建立非固相化SELEX技术, 最终获得识别甲胺磷的适体, 采用DNAMAN和RNA structure软件对适体进行一、二级结构分析。结果 经过10 轮筛选, ssDNA 文库与甲胺磷的亲和力呈上升趋势, 随机挑选的20个阳性克隆适体根据一级结构的同源性可分为5个家族, 二级结构预测以茎环结构为主。结论 本方法大大提高了适体的筛选效率, 最终筛选得到能识别甲胺磷的DNA适体。

英文摘要:

      Objective To select aptamers against methamidophos. Methods The ssDNA pool which was synthesized in vitro was immoboilized on surface of agarose gel. The ssDNAs that bound methamidophos were released from surface of agarose gel. Methamidophos aptamers were selected by non-solid phase SELEX. The primary and secondary structure of methamidophos aptamers were analysed by DNAMAN and RNA structure software. Results Affinity between ssDNA pool and methamidophos was increased after 10 selection rounds. The 20 ssDNAs which were positive clones were divided into five groups based on primary sequence homology. Hairpin loop was the main motif in the predicted secondary structure and it was supposed to be the binding part of the aptamers to methamidophos. Conclusion The efficiency of screening of the aptamer was greatly improved using this method and the methamidophos-specific DNA aptamers were selected successfully.