荧光PCR和数字PCR法检测转基因DAS-44406-6品系大豆

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。

Detection of Genetically Modified Soybean Event DAS-44406-6 by Real-Time PCR Method and Digital PCR Method

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.