【作者】 刘伟；

【导师】 田益玲； 张伟； 闫华光；

【作者基本信息】 河北农业大学 ， 生物与医药硕士（专业学位）， 2022， 硕士

【摘要】 赭曲霉毒素A(OchratoxinA,OTA)是一种由曲霉属和青霉属产生的真菌毒素,是污染食物的主要真菌毒素之一,对人类健康造成危害。因此,建立一种快速、准确地检测食品中OTA的方法,对保障食品安全和人体健康具有重要意义。本研究将适配体、链置换扩增技术(Strand displacement amplification,SDA)和电化学生物传感器进行联用构建了一种基于链置换扩增的电化学适配体生物传感器检测食品中OTA,主要研究内容如下:(1)根据OTA特异性适配体设计发卡结构,并在发卡结构的茎部设置SDA反应识别位点,进行SDA扩增,将扩增产物与修饰二茂铁(Ferrocene,Fc)的电化学探针进行杂交,使电信号发生变化,从而建立电化学适配体传感器检测OTA的方法。(2)本试验探究了发卡模板与OTA的孵育、SDA扩增以及电化学反应阶段的试验条件。得到最适的反应条件如下:发卡模板孵育时间为35 min;扩增体系中,Mg2+的添加量为1.5 μL,Nb.BsrDI切刻内切酶的酶活力浓度为1.00 U/μL,Bst DNA聚合酶的酶活力浓度为0.80 U/μL,扩增反应时间为30 min;电化学反应中,电化学探针修饰浓度为6 μmol/L,电化学探针孵育时间为4 h,电化学探针杂交时间为80 min。(3)在最适条件下,该传感器对OTA的线性检测范围为0.1 pg/mL～10 ng/mL,检测限为0.05 pg/mL,检出限为0.038 pg/mL;在特异性试验中,对OTA有较高的ΔI%,对于非OTA没有明显信号变化,说明该方法特异性良好;8次重复性试验结果表明,本研究具有稳定的电信号输出,相对标准偏差(RSD)为2.63%;在稳定性试验中,将制备电极放置14天后仍保持初始ΔI%的91.51%,稳定性较好;在人工加标样品试验中,结果表明加标回收率在96.60%～99.04%之间,与国标(GB5009.96-2016)中的酶联免疫吸附试验相比,检测结果具有良好的准确性。综上所述,本研究基于适配体和SDA建立了一种检测食品中OTA的电化学适配体生物传感器,该方法具有灵敏度高、特异性强、稳定性好等优点,且还可利用电流信号变化实现定量检测,结果准确,为食品中OTA的检测提供了新方法,具有良好的实际应用价值。更多还原

【Abstract】 Ochratoxin A(OTA)is a mycotoxin produced by Aspergillus and Penicillium,which is one of the main mycotoxins that contaminate food,causing harm to human health.Therefore,it is of great significance to establish a rapid and accurate method to detect OTA in foods for ensuring food safety and human health.In this study,an electrochemical aptamer biosensor based on strand displacement amplification(SDA)and electrochemical biosensor was constructed for the detection of OTA in food by combining aptamer,strand displacement amplification(SDA)and electrochemical biosensor.The main research contents were as follows:(1)The hairpin structure was designed according to the OTA-specific aptamer,and the SDA reaction recognition site was set in the stem of the hairpin structure,SDA amplification was performed,and the amplification product was hybridized with an electrochemical probe modified with Ferrocene(Fc)to change the electrical signal,thus establishing an electrochemical aptamer sensor for the detection of OTA.(2)In this experiment,the experimental conditions for the incubation of the hairpin template with OTA,the amplification of SDA,and the electrochemical reaction stage were explored.The optimal reaction conditions were as follows:incubation time of hairpin template was 35 min;In the amplification system,the addition of Mg2+was 1.5 μL,the enzyme activity concentration of Nb.BsrDI nicking endonuclease was 1.00 U/μL,the enzyme activity concentration of Bst DNA polymerase was 0.80 U/μL,and the amplification reaction time was 30 min;In the electrochemical reaction,the concentration of modification of electrochemical probes was 6 μmol/L.The incubation time of electrochemical probes was 4 h,and the hybridization time of electrochemical probes was 80 min.(3)Under optimal conditions,the linear detection range of the sensor for OTA was 0.1 pg/mL-10 ng/mL,and the LOD was 0.05 pg/mL,and the detection limit was 0.038 pg/mL.In the specificity test,the ΔI%for OTA was higher,but no significant signal change for non-OTA,indicating that the specificity of the method was good.The results of eight repeated experiments showed that the system has stable electrical signal output with a relative standard deviation of 2.63%.In the stability test,the prepared electrode remained 91.51%of the initial ΔI%after 14 days,showing good stability.In the artificial spiked sample test,the results showed that the spiked recovery was in the range of 96.60%～99.04%,according to the national standard(GB 5009.96-2016),the test results had good accuracy.In summary,in this study,we established an electrochemical aptamer biosensor for the detection of OTA in food based on aptamer and SDA.The method has the advantages of high sensitivity,well specificity,excellent stability.By using the change of current signal,this method can also achieve accurate quantitative detection,which provides an alternative for the detection of OTA in food with positive practical application value.更多还原