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

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。

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.