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| 转基因大豆‘ZH 10-6’数字PCR精准定量检测方法的建立 |
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刘双1,赵新1,李瑞环1,刘娜1,兰青阔1,檀建新2,王永1,2*
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| (1.天津市农业科学院 生物技术研究所, 天津 300381;2.河北农业大学 食品科技学院, 河北 保定 071001) |
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| 摘要: |
| 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R2>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。 |
| 关键词: 微滴式数字PCR 转基因耐除草剂大豆‘ZH 10-6’ 精准定量 转化体特异性 |
| DOI：10.11841/j.issn.1007-4333.2021.11.05 |
| 投稿时间：2020-12-03 |
| 基金项目:转基因重大专项(2019ZX08013-002,2019ZX08004001) |
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| Establishment of digital PCR method for quantitative detection of genetically modified soybean ‘ZH10-6' |
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LIU Shuang1,ZHAO Xin1,LI Ruihuan1,LIU Na1,LAN Qingkuo1,TAN Jianxin2,WANG Yong1,2*
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| (1.Biotechnology Research Institute, Tianjin Academy of Agricultural Sciences, Tianjin 300381, China;2.College of Food Science and Technology, Agricultural University of Hebei, Baoding 071001, China) |
| Abstract: |
| 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 R2>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. |
| Key words: droplet digital PCR genetically modified soybean ‘ZH 10-6' event specific transformant specificity |
