一种突变型循环肿瘤DNA荧光检测方法的构建

Construction of A Fluorescent Detection Method for Mutant Circulating Tumor DNA

首先合成二氧化硅包覆Fe_(3)O_(4)磁性纳米粒子(Fe_(3)O_(4)-SiO_(2)),然后连接目标物T790M的识别核酸链(DNA1),制备T790M的捕获探针(Fe_(3)O_(4)-SiO_(2)-DNA1)。进一步加入荧光素FAM标记的DNA2探针。当T790M存在时,DNA2,Fe_(3)O_(4)-SiO_(2)-DNA1与T790M发生DNA杂交。磁性分离后,上清液中DNA2上连接的FAM的荧光强度显著下降,且荧光强度与T790M浓度之间具有良好的线性相关性,线性回归方程为F=-142.56lgC+739.24(R^(2)=0.994),检测限为0.02 nmol/L。本方法为恶性肿瘤的精确诊断提供了一种有效的技术。

A capture probe for T790M(Fe_(3)O_(4)-SiO_(2)-DNA1)was firstly prepared by synthesizing silica-coated Fe_(3)O_(4)magnetic nanoparticles(Fe_(3)O_(4)-SiO_(2))and attaching the recognition nucleic acid strand(DNA1)of the target T790M.A fluorescein FAM-labelled DNA2 probe was added to the above probe.When T790M was presented,DNA2 and Fe_(3)O_(4)-SiO_(2)-DNA1 underwent DNA complementary hybridization with T790M.After magnetic separation,the fluorescence intensity of FAM attached on DNA2 in the supernatant was significantly reduced,and there was a good linear correlation between fluorescence intensity and T790M concentration,with a linear regression equation of F=-142.56lgC+739.24(R^(2)=0.994)and a detection limit of 0.02 nmol∙L^(-1).The fluorescence assay constructed in this work provided an effective technique for the accurate diagnosis of malignant tumors.