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| 荧光PCR和数字PCR法检测转基因DAS-44406-6品系大豆 | |
| 引用本文： | 于晓帆,高宏伟,孙 敏,肖西志,李荣贵. 荧光PCR和数字PCR法检测转基因DAS-44406-6品系大豆[J]. 食品科学, 2016, 37(16): 235-241. DOI: 10.7506/spkx1002-6630-201616038 |
| 作者姓名： | 于晓帆 高宏伟 孙 敏 肖西志 李荣贵 |
| 作者单位： | 1.青岛大学生命科学学院，山东 青岛 266071；2.山东出入境检验检疫局检验检疫技术中心，山东 青岛 266002 |
| 基金项目： | 公益性行业（质检）科研专项（201410014） |
| 摘 要： | 目的：建立实时荧光聚合酶链式反应（polymerase chain reaction，PCR）检测转基因DAS-44406-6品系大豆的定性检测方法和使用数字PCR检测转基因DAS-44406-6品系大豆的定量检测方法。方法：针对转基因DAS-44406-6大豆品系，进行5’-RACE，测定该品系转基因大豆外源片段与大豆染色体重组的边界序列，并根据该边界序列设计引物和探针。使用23 种非DAS-44406-6品系转基因植物作为阴性对照测试实时荧光PCR引物和探针的特异性，以DAS-44406-6品系样品制备6 个含量梯度的样品进行检测低限实验。使用数字PCR技术进行定量检测，并确定定量检测的低限。结果：建立的转基因DAS-44406-6大豆品系的实时荧光PCR特异性检测方法品系鉴定特异性较强，实时荧光PCR检测方法的检测低限在模板DNA浓度为100 ng/反应时，为0.01%的转基因大豆含量，约为16.6 个拷贝的DAS-44406-6基因组DNA；数字PCR检测方法的检测低限在模板DNA浓度为0.5 ng/反应、转基因大豆含量为1%时，相对标准偏差为0.7%。因此，建立的转基因DAS-44406-6大豆品系实时荧光PCR和数字PCR特异性检测方法符合转基因检测的要求。 |
| 关 键 词： | 转基因大豆 DAS-44406-6品系 品系鉴定 实时荧光PCR 数字PCR |
Detection of Genetically Modified Soybean Event DAS-44406-6 by Real-Time PCR Method and Digital PCR Method |
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| YU Xiaofan,GAO Hongwei,SUN Min,XIAO Xizhi,LI Ronggui. Detection of Genetically Modified Soybean Event DAS-44406-6 by Real-Time PCR Method and Digital PCR Method[J]. Food Science, 2016, 37(16): 235-241. DOI: 10.7506/spkx1002-6630-201616038 | |
| Authors: | YU Xiaofan GAO Hongwei SUN Min XIAO Xizhi LI Ronggui |
| Affiliation: | 1. College of Life Sciences, Qingdao University, Qingdao 266071, China; 2. Center of Inspection and Quarantine Technology,Shandong Entry-Exit Inspection and Quarantine Bureau, Qingdao 266002, China |
| Abstract: | Purpose: To determine the flanking sequence between exogenous and endogenous fragments using rapid amplification of cDNA ends (5’-RACE) and consequently to examine genetically modified soybean (GMS) DAS-44406- 6. Methods: Specific primers and probes were designed based on the flanking sequences of exogenous fragments of DAS- 44406-6. The specificity of the developed method was validated by using it to detect a variety of other GM samples and non- GM samples. DAS-44406-6 was used to prepare 6 content gradients to test the sensitivity of the method. At last, digital PCR was applied to measure nucleic acid molecules for absolute quantification. Conclusions: A real-time PCR method has been established for the identification of GMS soybean DAS-44406-6 with high specificity. The limit of detection (LODs) of the real-time PCR method at template DNA concentration of 100 ng/reaction and 0.01% GM soybean content was 16.6 copies of genomic DNA from DAS-44406-6. As for the LOD of the digital PCR method, the relative standard deviation (RSD) was 0.7% at template DNA concentration of 0.5 ng/reaction. The highly specific method combining real-time PCR and digital PCR could meet the requirements for the detection of GMS DAS-44406-6. |
| Keywords: | genetically modified soybean DAS-44406-6 event event-specific detection real-time PCR digital PCR |
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