发卡探针介导等温扩增荧光适体传感器检测玉米赤酶烯酮
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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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Detection of ZEN Using Hairpin Probe Mediated Isothermal Amplification of Fluorescent Aptamer Sensor
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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.College of Science and Technology, Hebei Agricultural University, Cangzhou 061100, Hebei;4.College of Life Sciences, Hebei Agricultural University, Baoding 071000, Hebei;5.Hebei Provincial Key Laboratory of Analysis and Control for Zoonoses Microbial, Baoding 071000, Hebei)

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    摘要:

    玉米赤酶烯酮(ZEN)主要由禾谷镰刀菌产生,广泛存在于小麦、大豆等谷物及其衍生产品中,具有雌激素作用和多种毒性,可造成神经系统的亢奋。本研究基于发卡探针介导等温扩增(HIAmp)的荧光适体传感器,定量检测食品中的ZEN。首先依据ZEN适体序列设计新型发卡探针,其次利用ZEN与适体特异性结合的特性,打开发卡结构启动扩增反应,最后结合荧光技术建立定量检测ZEN的方法,并对该方法进行评价。结果表明,在最优反应条件下,约1 h即可完成检测,检出限可达0.2 pg/mL,-lgCZEN与Ct值构建的线性方程为Ct = -1.195lgCZEN + 14.082(R2 = 0.9975),线性关系良好。特异性分析表明,仅存在ZEN的两组样品产生信号,其余5组不含有ZEN的样品皆未产生信号。加标回收率显示,此方法所得回收率为93.4%~99.6%,高于液相色谱法(92.5%~99.0%)和ELISA试剂盒方法(93.1%~98.4%)的回收率。本研究构建的基于发卡探针介导等温扩增的荧光适体传感器具有特异性强、耗时短、操作简便、灵敏度高及准确度高的优点,其检出限远低于国标液相色谱法(5 μg/kg)和ELISA试剂盒方法(0.5 ng/mL)的检出限,为谷物食品中ZEN的定量检测提供了一个新策略,在食品安全检测领域具有良好的应用前景。

    Abstract:

    Zearalenone (ZEN) is mainly produced by Fusarium graminearum and widely exists in wheat, soybean and other grains and their derivatives. It has estrogen effect and a variety of toxicity, which can cause nervous system excitement. In this study, a fluorescence aptamer sensor based on Hairpin probe-mediated Isothermal Amplification (HIAmp) was established to detect ZEN in food. Firstly, a new hairpin probe was designed according to the ZEN aptamer sequence. Secondly, the hairpin structure was opened to initiate the amplification reaction by using the specific binding property of ZEN to the aptamer. Finally, a quantitative detection method of ZEN was established by combining with fluorescence technology, and the method was evaluated. The results showed that this method can complete the detection in about 1 h under the optimal reaction conditions, and the detection limit can reach 0.2 pg/mL. The linear equation constructed by -lgCZEN and Ct value was Ct = -1.195 lgCZEN + 14.082 (R2 = 0.9975), and the linear relationship was good. The results of specific analysis showed that only two groups of samples containing ZEN produced signals, and the other five groups of samples without ZEN did not produce signals. The recoveries obtained by this method were 93.4%-99.6%, higher than those obtained by liquid chromatography (92.5%-99.0%) and ELISA kit(93.1%-98.4%). In summary, the fluorescent aptamer sensor based on hairpin probe mediated isothermal amplification constructed in this study has the advantages of strong specificity, short time consuming, simple operation, high sensitivity and high accuracy, and its detection limit is much lower than those of national standard liquid chromatography (5 μg/kg) and ELISA kit method (0.5 ng/mL). It provides a new strategy for the quantitative detection of ZEN in cereal foods and has a good application prospect in the field of food safety detection.

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佟欣,李子坤,张蕴哲,卢鑫,马晓燕,张伟.发卡探针介导等温扩增荧光适体传感器检测玉米赤酶烯酮[J].中国食品学报,2024,24(12):313-322

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  • 在线发布日期: 2025-01-23
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