基于杂交链式反应的新型荧光适体传感器检测食品中的OTA
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(1.河北农业大学食品科技学院 河北保定 071000;2.河北农业大学 河北保定 071000;3.河北农业大学理工系 河北沧州 061100;4.河北农业大学生命科学学院 河北保定 071000;5.河北省人畜共患病原微生物分析与防控重点实验室 河北保定 071000)

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国家自然科学基金项目(32172288,31371772);河北省自然科学基金重点项目(C2019204342);中央引导地方科技发展资金项目(216Z5501G,226Z5503G,236Z5502G);河北省外专百人计划项目(360-0803-JSN-3YGS);河北省博士后科研项目(B2021005007);河北省重点研究开发计划项目(18275501D);河北省教育厅科研项目(QN2022073);河北省自然科学基金项目(C2019204284)


A Novel Fluorescent Aptamer Sensor Based on Hybridization Chain Reaction for Detection of OTA in Food
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(1.College of Food Science and Technology, Hebei Agricultural University, Baoding 071000, Hebei;2.Hebei Agricultural University, Baoding 071000, Hebei;3.Department of Science and Technology, Hebei Agricultural University, Cangzhou 061100, Hebei;4.College of Life Science, Hebei Agricultural University, Baoding 071000, Hebei;5.Key Laboratory of Microbial Analysis and Prevention and Control of Zoonotic Diseases, Hebei Province,Baoding 071000, Hebei)

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    摘要:

    赭曲霉毒素A(OTA)是一种常见的霉菌毒素,常常污染食品,造成不同程度的危害。为开发一种基于杂交链式反应(HCR)的新型荧光适体传感器,用于快速灵敏地检测食品中的OTA,首先将OTA与适体结合,释放出触发链cDNA,进而触发发卡HP1与HP2发生HCR反应,使HP1中的G-四链体序列形成G-四链体结构,与此同时N-甲基中卟啉IX(NMM)染料插入到G-四链体结构中,从而显著增强荧光信号。通过荧光信号强度的变化,实现对OTA无酶、免荧光标记的定量检测。结果表明:本方法特异性强,在7组试验中,含有OTA的两组试验结果均是阳性,不含OTA的5组试验结果均为阴性。其线性回归方程为Y = 0.71133lgCOTA+3.21383,相关系数R2=0.9942,线性范围为0.1 pg/mL~5 ng/mL,检出限为0.1 pg/mL。将该方法应用于小麦、红酒和咖啡人工加标样品的检测,其回收率均在92%~97.88%之间,高于ELISA试剂盒的回收率(90.5%~95.78%),表明该方法的检测结果优于国标方法。本研究开发的新型荧光适体传感器可实现对食品中OTA的快速、灵敏、特异和准确地检测,也为其它毒素以及影响食品安全的其它化学污染物的快速检测提供了新思路。

    Abstract:

    Ochratoxin A (OTA) is a common mycotoxin that often contaminates food and causes different degrees of harm. In this study, a novel fluorescent aptamer sensor based on hybridization chain reaction (HCR) was developed for the rapid and sensitive detection of OTA in food products. Firstly, OTA binds to the aptamer and releases the trigger strand cDNA, which then triggers the HCR reaction between the hairpin HP1 and HP2, resulting in the formation of a G-quadruplex structure of G-quadruplex sequence in HP1, and at the same time, the N-quadruplex structure of G-quadruplex sequence in HP1. Sequence to form a G-quadruplex structure, while at the same time N-methylmesoporphyrin IX (NMM) dye is inserted into the G-quadruplex structure (G-quadruplex), which significantly enhances the fluorescence signal. The enzyme-free and fluorescence-free quantitative detection of OTA was achieved by the change of fluorescence signal intensity. The results showed that the method was highly specific, and among the seven groups of tests, the results of the two groups containing OTA were positive, and the results of the other five groups without OTA were negative. The linear regression equation was Y = 0.71133lgCOTA+3.21383 with the correlation coefficient R2=0.9942, the linear range was 0.1 pg/mL - 5 ng/mL, and the limit of detection was 0.1 pg/mL. The recoveries of this method applied to the detection of artificially spiked samples of wheat, red wine and coffee were all in the range of 92%-97.88%, which were higher than those of the recoveries of the ELISA kits (90.5%-95.78%) indicated that the method was superior to the national standard method. Therefore, the novel fluorescent aptasensor developed in this study can realize the rapid, sensitive, specific and accurate detection of OTA in food, and also provides a new idea for the rapid detection of other toxins as well as other chemical contaminants affecting food safety.

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贾耀博,张蕴哲,李子坤,卢鑫,徐慧,张伟.基于杂交链式反应的新型荧光适体传感器检测食品中的OTA[J].中国食品学报,2025,(1):391-399

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  • 收稿日期:2024-01-07
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  • 在线发布日期: 2025-03-05
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