激光诱导荧光结合磁分离检测CP4-EPSPS基因

doi:10.7506/spkx1002-6630-20200526-310

食品科学 ›› 2021, Vol. 42 ›› Issue (16): 218-223.doi: 10.7506/spkx1002-6630-20200526-310

激光诱导荧光结合磁分离检测CP4-EPSPS基因

庞月红，王逸盈，孙梦梦，沈晓芳，张毅

（江南大学食品学院，江苏无锡 214122）

发布日期:2021-08-27
基金资助:
国家自然科学基金面上项目（21976070；21876066）；中央高校基本科研业务费专项（JUSRP22003）

Magnetic Separation Combined with Laser Induced Fluorescence for Detection of CP4-EPSPS Gene

PANG Yuehong, WANG Yiying, SUN Mengmeng, SHEN Xiaofang, ZHANG Yi

(School of Food Science and Technology, Jiangnan University, Wuxi 214122, China)

Published:2021-08-27

摘要/Abstract

摘要： 建立一种激光诱导荧光结合磁分离的检测技术，用于在线检测CP4-5-烯醇丙酮酸酯-3-磷酸合酶基因（CP4-5-enol acetate-3-phosphate synthase gene，CP4-EPSPS）。以氨基磁纳米粒子（magnetic nanoparticles，MNPs）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-EPSPS（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质量浓度、捕获探针浓度、牛血清白蛋白质量浓度进行考察，在优化的实验条件下，在1.0×10－11～2.0×10－7?mol/L范围内，CP4-EPSPS基因浓度与荧光峰面积呈良好线性关系，检出限为4.0×10－12?mol/L（RSN=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: A laser-induced fluorescence (LIF) combined with magnetic separation sensing method was established for the online detection of the CP4-5-enol acetate-3-phosphate synthase gene (CP4-EPSPS). Amino magnetic nanoparticles (MNPs) were used as the vector to modify cDNA, construct gene capture probes, and design 5-carboxyfluorescein (FAM)-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 complementary base pairing. The construction could be enriched and separated by a magnetic field when passing through the capillary, and then its fluorescence signal could be detected online by a laser-induced fluorescence detector. At optimized concentrations of MNPs, cDNA and bovine serum protein, the concentration of the CP4-EPSPS gene in the range of 1.0 × 10-11–2.0 × 10-7 mol/L had a good linear relationship with the fluorescence peak area, and the limit of detection (LOD) was 4.0 × 10-12 mol/L (RSN = 3). This method could be successfully applied to the detection of polymerase chain reaction (PCR) amplification products of genetically modified soybeans.

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

中图分类号:

TS207.3

庞月红，王逸盈，孙梦梦，沈晓芳，张毅. 激光诱导荧光结合磁分离检测CP4-EPSPS基因[J]. 食品科学, 2021, 42(16): 218-223.

PANG Yuehong, WANG Yiying, SUN Mengmeng, SHEN Xiaofang, ZHANG Yi. Magnetic Separation Combined with Laser Induced Fluorescence for Detection of CP4-EPSPS Gene[J]. FOOD SCIENCE, 2021, 42(16): 218-223.

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激光诱导荧光结合磁分离检测CP4-EPSPS基因

Magnetic Separation Combined with Laser Induced Fluorescence for Detection of CP4-EPSPS Gene

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