食品科学 ›› 0, Vol. ›› Issue (): 0-0.

• 安全检测 •    下一篇

激光诱导荧光结合磁分离检测CP4-EPSPS基因

庞月红1,王逸盈2,3,孙梦梦2,3,沈晓芳3,张毅2,3   

  1. 1. 江南大学食品学院
    2.
    3. 江南大学
  • 收稿日期:2020-05-26 修回日期:2021-04-30 出版日期:2021-08-25 发布日期:2021-08-20
  • 通讯作者: 庞月红 E-mail:yhpang@jiangnan.edu.cn
  • 基金资助:
    国家自然科学基金;国家自然科学基金;中央高校基本科研业务费专项资金

Magnetic separation combined with laser induced fluorescence for detection of CP4-EPSPS gene

Yuehong Pang 2,2, 2,2,Xiao-fang Shen2, 2,2   

  • Received:2020-05-26 Revised:2021-04-30 Online:2021-08-25 Published:2021-08-20
  • Contact: Yuehong Pang E-mail:yhpang@jiangnan.edu.cn

摘要: CP4-5-烯醇丙酮酸酯-3-磷酸合酶基因(CP4-EPSPS)因其可以合成对除草剂草甘膦具有耐受性的5-烯醇丙酮酰-3-磷酸合成酶,是转基因大豆中使用最早,最普遍的抗除草剂性能的转基因。本文通过建立一种激光诱导荧光结合磁分离的在线检测技术,用于在线检测CP4-EPSPS基因。以氨基磁纳米粒子为载体修饰cDNA,构建捕获基因探针,并设计FAM荧光标记的信号基因探针(sDNA)。在目的基因CP4-EPSPE(tDNA)存在的情况下,cDNA和sDNA通过碱基互补配对原则与tDNA结合形成双链结构。该结构通过毛细管时被磁场富集分离,然后在激光诱导荧光检测器的作用下在线检测其荧光信号。通过对MNPs的浓度、捕获探针浓度、牛血清蛋白浓度进行考察,在优化的实验条件下,在1×10-11 mol/L~2×10-7 mol/L范围内,CP4-EPSPS基因的浓度与荧光峰面积成良好线性关系,检测限为4×10-12 mol/L(S/N=3),该方法成功应用于转基因大豆的聚合酶链式反应扩增产物的检测。

关键词: 激光诱导荧光, CP4-EPSPS基因, 转基因大豆

Abstract: The CP4-5-enol acetate-3-phosphate synthase gene (CP4-EPSPS) is the earliest and most commonly used herbicide-resistant transgene in genetically modified soybeans, because it can synthesize 5-enolpyruvyl-3-phosphate synthase that is resistant to the herbicide glyphosate. A laser-induced fluorescence and magnetic separation sensing method was established for the online detection of the CP4-EPSPS gene. Amino magnetic nanoparticles (MNPs) were used as vectors to modify cDNA, construct gene capture probes, and design FAM fluorescently labeled signal gene probes (sDNA). In the presence of the CP4-EPSPS gene probe(tDNA),cDNA and sDNA combined with tDNA to form a double-stranded structure through the principle of tandem complementary pairing. The construction could be enriched and separated by a magnetic field when it through the capillary, and then its fluorescence signal could be detected online under the action of a laser induced fluorescence detector. By optimizing the concentration of MNPs, the concentration of the cDNA and the concentration of bovine serum protein, it was found that under the optimal conditions, within the range of 1.0×10-11 mol/L~2.0×10-7 mol/L, the concentration of CP4-EPSPS gene has a good linear relationship with the fluorescence peak area, and the limit of detection (LOD) was 4.0×10-12 mol/L (S/N=3). This method could be successfully applied to the detection of polymerase chain reaction amplification products of genetically modified soybeans.

Key words: laser induced fluorescence, CP4-EPSPS gene, genetically modified soybean