转基因玉米双抗12-5荧光RPA现场可视化检测方法的建立

Establishment of a field visual detection method for genetically modified maize‘Shuangkang’12-5 by fluorescence RPA

转基因玉米双抗12-5具有良好的抗虫性和除草剂耐受性,是我国第一批获得安全证书的转基因玉米之一,具有广阔的应用前景。本研究利用重组酶聚合酶扩增技术(recombinase polymerase amplification,RPA)建立转基因玉米双抗12-5的现场快速检测方法。针对转基因玉米双抗12-5的转化体特异性序列片段,设计引物和探针,通过引物筛选实验得到最佳引物与探针组合。荧光RPA扩增结果可以在蓝光下直接进行可视化分析。结果表明,建立的转基因玉米双抗12-5可视化检测体系特异性强,检测灵敏度达到10拷贝。进一步研究发现RPA的扩增体系对温度有很强的适应性,样品在34℃~46℃之间都能得到扩增,据此,本研究利用市面上常见的自发热暖贴代替常规的加热仪器来激发RPA。结果表明,自发热暖贴满足RPA扩增体系对温度的需要。最终,本研究将自发热暖贴加热法与RPA可视化检测体系结合,对转基因玉米双抗12-5进行现场检测,并与qPCR方法检测结果作比较,检测结果表明,本研究建立的现场可视化检测方法与qPCR方法检测结果一致,并且可视化检测方法时间短,检测结果清晰易分辨。本研究建立的转基因玉米双抗12-5现场快速可视化检测方法,其特异性强、灵敏度高、方便、快捷,不仅满足了转基因玉米双抗12-5的现场快速检测的需要,也为其他现场快速检测方法的开发提供了新思路。

The genetically modified(GM)maize‘Shuangkang’12-5 has good insect resistance and herbicide tolerance,which is one of the first series of GM maizes obtained a safety certificate in China,and it has broad application prospect in the future.This study established an on-site rapid detection method for GM‘Shuangkang’12-5 based on recombinase polymerase amplification(RPA)technology,which primes and probe were designed according to the specific flank sequence.Then the best combination of primers and probe was obtained through a screeing process.The amplification results of fluorescence RPA can be directly visualized under blue light.The results showed that the visual detection system of GM‘Shuangkang’12-5 with high specificity,and the detection sensitivity of the method could reached 10 copies.Further research found that the RPA amplification system had a wide range of temperature(34℃-46℃).According to this property,the common self-heating warm pastes on the market were used replace the traditional heating instruments to stimulate the RPA.The results showed that the self-heating warm paste meets the temperature requirement of the RPA system.Finally,we combined the self-heating warm pastes with the RPA visual detection system to conduct on-site detection of GM‘Shuangkang’12-5,and compared the results with the detection results of qPCR.The detection showed that the results of on-site visual detection method established in this study were consistent with the detection results of the qPCR.Moreover,the visual detection method was more shorter in time and the final detection result was clear and easy to distinguish.The rapid on-site visual detection method for GM‘Shuangkang’12-5 established in this study has high specificity,high sensitivity and convenience.It not only meets the needs of on-site rapid detection of GM‘Shuangkang’12-5,but also provides highlight for the development of other on-site rapid detection methods.