基于分子信标荧光纳米探针的李斯特菌DNA均相检测方法

Homogenous Detection of Listeria monocytohenes DNA with Molecule Beacon and Highly Fluorescent Bioconjugated Nanoparticles Probe

基于分子信标(MB)识别和荧光纳米粒子探针技术,建立了均相体系中李斯特菌目标DNA的高灵敏检测新方法.首先以羊抗人免疫球蛋白(IgG)标记的异硫氰酸荧光素(FITC)为核材料,成功制备了FITC-IgG@SiO2核壳荧光纳米粒子,有效防止了传统方法中采用单一FITC制备纳米颗粒时泄露严重的问题.随后以FITC-IgG@SiO2荧光纳米粒子和纳米金分别标记单核细胞增生李斯特菌序列特异性分子信标探针5'端和3'端,成功构建了单核细胞增生李斯特菌序列特异性分子信标荧光纳米探针.在实验优化条件下,α(令α=F/F0,F代表MB和目标DNA杂交以后的荧光强度,F0代表MB完全闭合时的荧光强度)与目标DNA浓度在1～200pmol/L浓度范围内呈良好的线性关系,检出下限为0.3pmol/L,相对标准偏差为2.6%(50pmol/L,n=11).将该方法应用于食品样品中单核细胞增生李斯特菌的检测,结果与国标法一致.

Based on molecular beacon recognition and nanoparticles labeling technique, we constructed a sensitive detection method for target DNA of Listeria monocytogenes in homogeneous system. Using FITC-IgG complex as nuclear materials, FITC-IgG@SiO2 core/shell fluorescence nanoparticles were successfully synthesized. It effectively prevented the disclosure of FITC in traditional preparation process. Then the prepared fluorescence nanoparticles and Au nanoparticles were used to label 5’ and 3’ end of L. monocytogenes sequence-specific molecular beacon probe, respectively. The molecular beacon-fluorescent nanoparticles probe was successfully constructed. Under the optimized conditions, it was found that the fluorescent intensity is proportional to the concentration of target DNA of L. monocytogenes over the range of 1～200 pmol/L with a detection limit of 0.3 pmol/L （3S/N）, and the relative standard deviation is 2.6% （50 pmol/L, n=11）. The proposed method was applied to detect L. monocytogenes in food samples, the results obtained are identical to that of by an official standard method.