基于适体技术的桔青毒素检测方法的建立

桔青毒素（citrinin,CTN）是以玉米、谷物、奶酪等为主要成分的食品和动物饲料中常见的、由桔青霉菌产生的酮类真菌毒素,可引起人和动物的慢性中毒或癌症,一直缺少灵敏的快速检测方法。本文通过指数富集适体系统进化技术（简称SELEX）对可能与CTN结合的特异性适体进行了筛选,经过15轮循环,得到17条适体。通过二级结构分析、亲和力检测发现适体13（Apt-13）对CTN有较好的亲和度,解离常数Kd为0.06μmol/L。进一步利用非荧光标记染料PicoGreen,利用其与双链DNA结合的原理,建立了桔青毒素非标记荧光检测方法,30min完成检测,最低检测限达到国家标准（50ppb）且与其他毒素无交叉反应。本研究建立的基于适体的桔青毒素检测技术成本低,可以替代传统的基于抗体的检测方法,为霉菌毒素的精准检测技术的开发提供了实验证据。

Development of an aptamer based technique for the detection of mycotoxin citrinin

Citrinin (CTN), a tetraketide metabolite of Penicillium citrinum, is a common mycotoxin found in human foods and animal feeds (corn, grain, cheese etc), causing food poison or carcinogenesis to human and animals. For rapid detection of citrinin, accurate and reliable methods are in demand. In this study, specific aptamers against citrinin were selected by using the systematic evolution of ligands by exponential enrichment (SELEX) method. After 15 rounds of SELEX cycles, 17 aptamers were obtained. Of which, aptamer Apt-13 having a close affinity to CTN was further selected based on secondary structure analysis. The dissociation constant (Kd) of Apt-13 was 0.06µmol/L. An accurate and rapid CTN detection method was developed by further using Apt-13 and PicoGreen, a specific double-stranded DNA binding and non-labeled fluorescent dye. The detection procedure could be finished in 30min. The limit of detection (sensitivity) of this method was 50ppb, which could meet the national standard for CTN residue detection. No cross reactions were found to other mycotoxins. This aptamer-based method exhibited more advantages than traditional antibody-based methods due to its low cost, thus could be used as an alternative tool for mycotoxin detection.