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化学学报
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化学学报 ›› 2010, Vol. 68 ›› Issue (9): 909-916. 上一篇    下一篇

研究论文

基于分子信标荧光纳米探针的李斯特菌DNA均相检测方法

王周平，徐欢，段诺，吴佳，叶菁，乐国伟   

  1. (江南大学食品科学与技术国家重点实验室 江南大学食品学院 无锡 214122)
  • 投稿日期:2009-09-07 修回日期:2009-12-07 发布日期:2010-01-13
  • 通讯作者: 王周平 E-mail:wangzp1974@hotmail.com
  • 基金资助:

    国家“863”计划(2008AA10Z419)、国家自然科学基金(20805019)、教育部博士点基金新教师计划(20070295014)、江苏省自然科学基金(BK20081603)、“111”引智计划(111 project-B07029)、教育部创新团队计划(PCSIRT0627)

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

WANG Zhou-Beng, XU Huan, DUAN Nuo, TUN Jia, XIE Jing, LE Guo-Wei   

  1. (State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi 214122)
  • Received:2009-09-07 Revised:2009-12-07 Published:2010-01-13

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2103

被引次数

18 | 2

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1～200 pmol/L浓度范围内呈良好的线性关系, 检出下限为0.3 pmol/L, 相对标准偏差为2.6% (50 pmol/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.

关键词: FITC-IgG@SiO2荧光纳米粒子, 纳米金, 分子信标, 单核细胞增生李斯特菌, DNA杂交

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.

Key words: FITC-IgG@SiO2 fluorescent nanoparticle, gold nanoparticle, molecular beacon, Listeria monocytogenes, DNA hybridization