转基因大豆‘ZH 10-6’数字PCR精准定量检测方法的建立

Establishment of digital PCR method for quantitative detection of genetically modified soybean‘ZH10-6'

为实现转基因耐除草剂大豆品种‘ZH 10-6’转化体成分的精准定量检测,以转基因大豆‘ZH 10-6’的5′端边界序列为靶序列,设计PCR扩增引物和TaqMan探针,并对引物探针浓度进行优化,经特异性测试,建立了耐除草剂大豆品种‘ZH 10-6’的微滴式数字PCR检测方法。结果表明:1)特异性良好:只有以转基因耐除草剂大豆品种‘ZH 10-6’基因组为模板才有扩增信号;2)灵敏度高:在相对标准偏差≤25%的情况下,方法的检出限(LOD)为13.0个拷贝;定量限(LOQ)为66.0个拷贝;3)PCR扩增反应模板含量与样品拷贝数之间线性相关系数R^(2)>0.99,DNA含量线性范围为0.08%~100.00%。综上,本研究建立的转基因耐除草剂大豆‘ZH 10-6’转化体定量方法特异性强、稳定性好、准确度和灵敏度高,可用于对转基因大豆品种‘ZH 10-6’的定量检测。

In order to achieve the accurate quantitative detection of the composition in genetically modified herbicide-tolerant soybean variety‘ZH 10-6',5′flanking sequence of the genetically modified soybean‘ZH 10-6'was used as the target sequence,PCR amplification primers and TaqMan probes were designed,and the primer probe concentration was optimized.After specific testing,a droplet digital PCR detection method for the herbicide-tolerant soybean variety‘ZH 10-6'was established.The results show that:1)The specificity was good.Only when the genetically modified herbicide-tolerant soybean‘ZH 10-6'genome was used as a template,will there be amplification signals;2)The sensitivity was high.When the relative standard deviation was≤25%,the detection limit of this method(LOD)is 13 copies,and the limit of quantification(LOQ)was 66 copies;3)The results of PCR amplification reaction displayed a linear correlation coefficient between template content and sample copy number is R^(2)>0.99,The linear range of DNA content is 0.08%-100.00%.In conclusion,the quantitative detection method for transgenic soybean‘ZH 10-6'transformants established in this study has strong specificity,good stability,high accuracy and sensitivity,and can be used for the quantitative detection of genetically modified soybean‘ZH 10-6'strains quickly,simply and efficiently.