转基因大豆中外源基因g10evo-epsps的qPCR检测方法的建立和验证

Development and validation of qPCR method for detection of g10evo-epsps in genetically modified soybean

耐除草剂基因g10evo-epsps在我国转基因农作物研发中常常被用作目标性状基因或筛选标记基因,因此可作为转基因成分检测的重要筛查基因,但目前缺少相应的检测方法。本研究旨在建立g10evo-epsps基因特异性的实时荧光定量PCR(qPCR)检测方法,从而为转基因大豆的监测提供一种稳定可靠的方法体系。本研究基于转基因大豆中的g10evo-epsps基因序列设计了qPCR检测引物和探针,并对检测方法的特异性、灵敏度、准确度和精确性进行了实验室内验证。结果显示,建立的g10evo-epspsqPCR检测方法能够特异性检测转基因大豆ZUTS-33中的g10evo-epsps目的基因;标准曲线分析表明,3次重复试验的扩增效率在90%以上,R2均大于0.99;方法的检测限(LOD)为不高于8拷贝,定量限(LOQ)推测为16拷贝;对含量为4.5%、2%、0.5%、0.09%和0.045%的转基因大豆ZUTS-33基因组DNA进行准确度分析,发现该方法测定的平均值和预期值之间的偏差为0.00%~11.11%,3次重复试验的RSDr为2.30%~17.10%。结果表明本研究建立的g10evo-epsps qPCR检测方法能够满足转基因大豆筛查检测的要求,为转基因大豆监管和标识提供技术支撑。

Herbicide tolerant gene g10evo-epsps was usually used in genetically modified(GM)crops in China.To develop a detection method by specific quantitative PCR,g10evo-epsps-specific primers and probe were designed and used for accurate and precise real-time PCR(qPCR)method in house validated.Results supported the specific of the method on detecting g10evo-epsps gene from transgenic soybean ZUTS-33.Standard curves construction showed that the PCR amplification efficiency was over 90%with R2 above 0.99.Limit of detection was assessed to be 8 copies and limit of quantification was estimated at 16 copies.The accuracy of qPCR method was verified by screening 5 mixed DNA samples with known levels of the ZUTS-33 event(4.5,2,0.5,0.09 and 0.045%,respectively).The average bias between the quantified values and expected values of the 5 samples deviated from 0.00%to 11.11%,and relative repeatability standard deviation ranged from 2.30%to 17.10%.The overall data indicated that this developed g10evo-epsps-specific qPCR method could meet the requirements of GM soybean screening and provide technical support for GM soybean regulation and labeling.