Ministry of Education, Key Laboratory of Analysis and Detection for Food Safety, Fujian Provincial Key Laboratory of Analysis and Detection for Food Safety, Department of Chemistry, Fuzhou University, Fuzhou, 350002, China.
Anal Chem. 2009 Dec 1;81(23):9578-84. doi: 10.1021/ac901510s.
A capillary electrophoresis coupled with electrochemiluminescent detection system (CE-ECL) was developed for the detection of polymerase chain reaction (PCR) amplicons. The ECL luminophore, tris(1,10-phenanthroline) ruthenium(II) (Ru(phen)(3)(2+)), was labeled to the PCR primers before amplification. Ru(phen)(3)(2+) was then introduced to PCR amplicons by PCR amplification. Eventually, the PCR amplicons were separated and detected by the homemade CE-ECL system. The detection of a typical genetically modified organism (GMO), Roundup Ready Soy (RRS), was shown as an example to demonstrate the reliability of the proposed approach. Four pairs of primers were amplified by multiple PCR (MPCR) simultaneously, three of which were targeted on the specific sequence of exogenous genes of RRS, and another was targeted on the endogenous reference gene of soybean. Both the conditions for PCR amplification and CE-ECL separation and detection were investigated in detail. Results showed that, under the optimal conditions, the proposed method can accurately identifying RRS. The corresponding limit of detection (LOD) was below 0.01% with 35 PCR cycles.
建立了毛细管电泳电化学发光检测（CE-ECL）系统，用于聚合酶链反应（PCR）扩增子的检测。电化学发光标记物三（1,10-菲啰啉）钌（II）（Ru（phen）（3）（2+））在扩增前标记到 PCR 引物上。然后，通过 PCR 扩增将 Ru（phen）（3）（2+）引入到 PCR 扩增子中。最后，通过自制的 CE-ECL 系统对 PCR 扩增子进行分离和检测。以 Roundup Ready Soy（RRS）为例，展示了该方法的可靠性。通过多重 PCR（MPCR）同时扩增了四对引物，其中三对针对 RRS 外源基因的特定序列，另一对针对大豆的内参基因。详细研究了 PCR 扩增和 CE-ECL 分离检测的条件。结果表明，在最佳条件下，该方法可以准确鉴定 RRS。在 35 个 PCR 循环下，对应的检测限（LOD）低于 0.01%。
