| 候吉超 1, 2, 李忠鹏 1, 梁雨欣 1, 张春雨 1, 李小宇 1*, 王永志 1*.转 Cp4 epsps 基因大豆快速DAS-ELISA 检测方法的建立[J].植物保护,2021,47(4):128-133. |
| 转 Cp4 epsps 基因大豆快速DAS-ELISA 检测方法的建立 |
| Establishment of rapid DAS-ELISA for detection of the Cp4 epsps transgenic soybean |
| 投稿时间：2020-03-17 修订日期：2020-05-06 |
| DOI：10.16688/j.zwbh.2020133 |
| 中文关键词: CP4-EPSPS 双抗夹心ELISA 大豆 检测 |
| 英文关键词:CP4-EPSPS double antibody sandwich ELISA (DAS-ELISA) soybean detection |
| 基金项目:吉林省省级产业创新专项资金(2017C057-1); 吉林省科技厅重点研发项目(20180201084SF):转基因农产品快速检测试剂的研发与应用 |
|
| 摘要点击次数: 480 |
| 全文下载次数: 290 |
| 中文摘要: |
| 为了快速检测转 Cp4 epsps 基因大豆, 本研究以纯化后的CP4-EPSPS单克隆抗体1D12为捕获抗体, 辣根过氧化物酶标记的羊抗CP4-EPSPS多克隆抗体8092为检测抗体, 通过优化抗体工作浓度和抗原抗体反应时间, 成功建立转 Cp4 epsps 基因大豆快速双抗夹心ELISA检测方法?结果显示:最佳检测条件为捕获抗体浓度10 μg/mL, 检测抗体浓度1.25 μg/mL, 样品与检测抗体先后加入酶标板37℃共同孵育60 min?该方法的检测范围为0.312 5~80 μg/mL, 待检叶片?籽粒最佳检测范围为10~80倍稀释; 板内变异系数为1.64%~5.42%, 板间变异系数为3.05%~9.13%, 符合ELISA定性试剂盒参考标准; 对100份大豆叶片?20份大豆籽粒进行检测, 与Western blot结果和标准结果进行比较, 符合率为100%, 表明该检测方法具有良好的准确性和重复性?该检测方法75 min内即可完成检测, 适用于快速检测转 Cp4 epsps 基因大豆, 为转 Cp4 epsps 基因快速检测试剂盒的开发奠定了基础? |
| 英文摘要: |
| The purified anti-CP4-EPSPS monoclonal antibody 1D12 was used as a capture antibody and the sheep anti-CP4-EPSPS polyclonal antibody 8092 conjugated horseradish peroxidase was used as the detection antibody to rapidly detect the Cp4 epsps transgenic soybean in this study. The rapid double antibody sandwich ELISA (DAS-ELISA) detection method for detecting Cp4 epsps transgenic soybean was successfully established by optimizing antibody working concentration and antigen-antibody reaction time. The results showed that the optimal detection conditions were 10 μg/mL of the capture antibody and 1.25 μg/mL of the detection antibody, with the sample and detection antibody co-incubated in the ELISA plate for 60 min at 37℃. The detection range of CP4-EPSPS protein was 0.312 5 -80 μg/mL. The optimal dilution rate for sample soybean leaves and seeds were 10-80 times. The intraplate variation coefficient was 1.64%-5.42%, and the interplate variation coefficient was 3.05%-9.13%. These results met the ELISA qualitative kit standard. A total of 100 soybean leaves and 20 soybean seeds were detected by rapid DAS-ELISA and Western blot. The consistency rate was 100%, with a good accuracy and repeatability. This testing program could be completed within 75 min. It was suitable for rapid detection of Cp4 epsps transgenic soybean and laid a foundation for the development of rapid detection kit for Cp4 epsps gene. |
| 查看全文 查看/发表评论 下载PDF阅读器 |
| 关闭 |
|
|
|
