黄曲霉毒素B1核酸适配体筛选和检测方法的建立  

Screening of Aptamers for Aflatoxin B1 and Establishment of a Method for Aflatoxin B1 Detection

作　　者：张敏[1] 张先舟[1] 李聪[1] 高浩 刘健慧 田益玲[1] 马雯[1] 李英军[1] 檀建新[1] ZHANG Min;ZHANG Xianzhou;LI Cong;GAO Hao;LIU Jianhui;TIAN Yiling;MA Wen;LI Yingjun;TAN Jianxin(Engineering Research Center of Hebei Province for Agricultural Products Processing,College of Food Science and Technology,Hebei Agricultural University,Baoding 071000,China)

机构地区：[1]河北农业大学食品科技学院,河北省农产品加工工程技术中心,河北保定071000

出　　处：《食品科学》2020年第24期295-303,共9页Food Science

基　　金：“十三五”国家重点研发计划重点专项(2018YFD0400200);河北省重点研发计划项目(16275505D);河北省食品科学与工程学科“双一流”建设资金项目(2016SPGCA18)。

摘　　要：建立一种高灵敏的快速检测黄曲霉毒素B1(aflatoxin B1,AFB1)的方法。基于氧化石墨烯(graphene oxide,GO)通过π-π共轭吸附单链寡核苷酸(single-strand DNA,ssDNA),对已结合靶标的ssDNA吸附能力较弱的特性,设计针对AFB1核酸适配体的筛选方案。经过10轮筛选,共获得45条ssDNA序列。挑选具有代表性的6条序列进行亲和力分析实验,其解离常数Kd分别为16.29、27.88、18.54、23.11、47.94、22.31 nmol/L。选用亲和力较高的AFB-5、AFB-8进行特异性实验,结果显示所筛选的适配体能特异性吸附AFB1。最后利用适配体AFB-8作为识别元件建立了基于纳米金比色检测AFB1的方法。在优化的检测条件下,AFB1质量浓度与A520 nm值有良好的线性关系,其回归方程为y=-0.00709x+0.46142(R^2=0.9979),线性范围为0.025~10 ng/mL,检出限为0.025 ng/mL。通过特异性实验和对大米样品加标回收实验,加标回收率为83.36%~105.74%,证明本方法的可行性。In this paper,we aimed to develop a sensitive and rapid method to detect aflatoxin B1(AFB1).Based on the fact that graphene oxide(GO)can efficiently adsorb single-strand oligonucleotides(ssDNA)throughπ-πconjugation,but hardly adsorb ssDNAs bound to the targets,a scheme for screening aptamers for AFB1 was designed and performed.After 10 rounds of screening,a total of 45 ssDNA sequences were obtained.Secondly,six representative sequences were selected for further affinity analysis,whose dissociation constants(Kd)were 16.29,27.88,18.54,23.11,47.94,and 22.31 nmol/L,respectively.Subsequently,the specificity of the ssDNAs AFB-5 and AFB-8,with higher affinity,were determined.The results revealed that the selected aptamers could bind to AFB1 with high specificity.Finally,AFB-8 was used as recognition element to construct a gold nanoparticles(AuNPs)-based colorimetric assay for AFB1。Under the optimized detection conditions,AFB1 concentration(x)showed a good linear relationship with A520 nm(y),which was expressed by the following equation:y=−0.00709x+0.46142(R^2=0.9979).The linear range and the detection limit were 0.025–10 ng/mL and 0.025 ng/mL,respectively.The specificity of this method was evaluated and the recoveries for spiked rice samples were in the range of 83.36%–105.74%.The results obtained proved the feasibility of the method.

关 键 词：黄曲霉毒素B1 核酸适配体 指数富集的配体系统进化技术 比色检测 纳米金 

分 类 号：TS201.6[轻工技术与工程—食品科学]