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中国农业科学院奶产品质量安全与风险评估创新团队研究建立了牛奶黄曲霉毒素M1(AFM1)的核酸适配体检测新方法

来源:中国农业科学院 新闻中心 http://www.caas.cn/xwzx/kyjz/313710.html发布时间:2021-07-25

黄曲霉毒素检测

牛奶黄曲霉毒素M1(AFM1)的核酸适配体检测方法

Aptamer-Based Fluorescence Quenching Approach for Detection of Aflatoxin M1 in Milk

       中国农业科学院北京畜牧兽医研究所奶产品质量安全与风险评估创新团队研究建立了牛奶黄曲霉毒素M1(AFM1)的核酸适配体检测新方法。
       该方法具有灵敏度高、准确度高、操作简便等优点,为快速检测牛奶中的AFM1开辟了新路径。相关研究成果发表在《化学前沿(Frontiers in Chemistry)》上。
       
据团队执行首席郑楠研究员介绍,AFM1是牛奶中最重要的霉菌毒素,目前AFM1常用检测方法有HPLC、LC-MS/MS等,但这些方法对样品预处理要求高,且耗费时间长。

       为进一步提高样品检测的质量和效率,研究人员经过长期摸索和反复验证,成功建立了利用核酸适配体技术检测牛奶AFM1的新方法。
       研究表明,在核酸适配体传感检测中,AFM1与反应荧光强度呈线性关系,线性范围为1纳克/毫升~100纳克/毫升,AFM1检测限为0.5纳克/毫升,加标回收率可达到93.4%~101.3%。

  该研究得到中国农科院创新工程项目支持。

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FIGURE 1. (A) Schematic diagram of the aptamer-based fluorescence quenching platform for the detection of AFM1. (B) Fluorescence emission spectra of the aptamer-based sensing system: blank (10 mM Tris-HCl buffer, pH 7.0) (A), AFM1 aptamer hybridized with cDNA (B), 150 ng/mL AFM1(C), and AFM1 aptamer alone (D). Excitation/emission λex/λem = 495/520 nm. Reaction conditions: 10 nM AFM1 aptamer and 20 nM cDNA in Tris-HCl buffer (10 mM, pH 7.0).
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FIGURE 2. Optimization of cDNA sequence, concentration, and reaction stability.
(A) Optimization of cDNA by varying its length (cDNAs 1–7). Black bars represent fluorescence intensity before addition of AFM1, and blue bars represent the fluorescence intensity after addition of 150 ng/mL AFM1. (B) Fold increase in fluorescence (F/F0) after addition of 150 ng/mL AFM1. (C) Optimization of the aptamer:cDNA5 concentration ratio in the presence of 150 ng/mL AFM1. (D) Study on the stability of AFM1 fluorescence sensor after addition of 150 ng/mL AFM1. Data represent means and standard deviations from three parallel experiments. Excitation/emission: λex/λem = 495/520 nm. Reaction conditions: 10 nM AFM1 aptamer and 20 nM cDNA in Tris-HCl buffer (10 mM, pH 7.0).
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FIGURE 3. (A) Fluorescence emission spectra of the aptasensor. (B) Linear relationship between fluorescence intensity and AFM1 concentration. Data represent means and standard deviations from three parallel experiments. Excitation/emission: λex/λem = 495/520 nm. Reaction conditions: 10 nM AFM1 aptamer and 20 nM cDNA in Tris-HCl buffer (10 mM, pH 7.0).
TABLE 1. Comparison of the aptasensor with previously reported methods for detection of AFM1.
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FIGURE 4. Fluorescence intensity in the absence (control) and presence of 40 ng/mL mycotoxins: AFM1, AFB1, AFB2, AFG1, AFG2, OTA, ZEN, and FB1, MIX1 (AFB1, AFB2, AFG1, AFG2, OTA, ZEN, and FB1), and MIX2 (AFM1, AFB1, AFB2, AFG1, AFG2, OTA, ZEN, and FB1). Excitation/emission λex/λem = 495/520 nm. Reaction conditions: 10 nM AFM1 aptamer and 20 nm cDNA in Tris-HCl buffer (10 mM, pH 7.0).
TABLE 2. Determination of AFM1 spiked into milk samples (n = 5).
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信息来源:http://www.caas.cn/xwzx/kyjz/313710.html
论文链接:https://www.frontiersin.org/articles/10.3389/fchem.2021.653869/full

 

文稿/编辑:上官国莲        审核:刘阳