副溶血性弧菌特异性核酸适配体筛选  

SELEX of Vibrio parahaemolyticus Specific Aptamer

作　　者：李洁 岳晓禹[2] 崔丽伟[2] 许文涛 李相阳[1] LI Jie;YUE Xiaoyu;CUI Liwei;XU Wentao;LI Xiangyang(Faculty of Food Science and Engineering,Beijing University of Agriculture,Beijing 102206,China;College of Food and Bioengineering,Henan Institute of Animal Husbandry,Zhengzhou 450046,China;Department of Nutrition and Health,Institute of Nutrition and Health,China Agricultural University,Beijing 100083,China)

机构地区：[1]北京农学院食品科学与工程学院,北京102206 [2]河南牧业经济学院食品与生物学院,郑州450046 [3]中国农业大学营养与健康系,北京100083

出　　处：《生物技术进展》2023年第4期612-618,共7页Current Biotechnology

基　　金：北京市景观休闲农业创新团队项目(BAIC09-2022)。

摘　　要：副溶血性弧菌是水产品中常见的食源性致病菌,其快速检测方法逐渐被开发,适配体作为一种新兴的特异性识别元件受到广泛关注。以副溶血性弧菌作为靶标菌进行了12轮细胞-指数富集配体系统进化技术(cell-systematic evolution of ligands by exponential enrichment,cell-SELEX),其中包括9轮正向筛选和3轮负向筛选,在每轮正筛中对PCR轮数和Lambada外切酶用量进行优化,筛选完成后进行核酸测序,并通过qPCR和流式细胞术多角度地对测序得到的序列性能进行验证。结果显示,F-28-10表现出较好的结合能力和特异性,且通过对PCR扩增轮数和Lambda外切酶用量优化后的cell-SELEX可以实现微生物特异性适配体的开发。研究结果为食源性致病微生物快速检测技术的开发提供了新的可利用的识别元件。As a common foodborne pathogenic bacterium in aquatic products,and a variety of rapid detection methods of Vibrio parahaemolyticus have been gradually developed.Aptamers have received widespread attention as an emerging specific identification element.In this study,12 rounds of cell-systematic evolution of ligands by exponential enrichment(cell-SELEX)were performed with Vibrio parahaemolyticus as the target,including 9 rounds of positive selection and 3 rounds of negative selection,and the number of PCR rounds and the dosage of Lambada exonuclease were optimized in each round of positive selection.Nucleic acid sequencing was performed after SELEX,and the obtained sequence performance was verified from multiple angles by qPCR and flow cytometry.The results showed that F-28-10 showed better binding ability and specificity.And the development of aptamers can be achieved by optimizing the number of PCR amplification rounds and the amount of Lambda exonuclease in cell-SELEX.This method provided a new usable identification element for the development of rapid detection technology of foodborne pathogenic microorganisms.

关 键 词：副溶血性弧菌 适配体 SELEX 

分 类 号：TS207.4[轻工技术与工程—食品科学] R155.5[轻工技术与工程—食品科学与工程]