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| 基于焦磷酸测序技术的基因编辑位点检测方法的建立 |
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李葱葱1, 高越2,3, 沈晓玲2, 李飞武1, 赵新2, 龙丽坤1, 李亮4, 王永2, 兰青阔2
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| (1.吉林省农业科学院 农业质量标准与检测技术研究所, 长春 130033;2.天津市农业质量标准与检测技术研究所, 天津 300381;3.河北农业大学 植物保护学院, 河北 保定 071000;4.中国农业科学院 生物技术研究所, 北京 100081) |
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| 摘要: |
| 为探索建立基因编辑农产品的检测方法，本研究以猪MSTN基因定点编辑位点为靶标，通过设计PCR-焦磷酸测序引物、优化测序碱基次序、构建标准曲线等步骤，建立基于焦磷酸测序技术的基因编辑产品定性和定量检测方法。结果表明，该方法能够准确检测出MSTN基因第3外显子的AG缺失位点，具备基因分型定性判定和等位基因频率定量检测的功能，从而准确检测样品是否为MSTN基因编辑猪产品，以及样品基因编辑产品的含量。 |
| 关键词: 基因编辑猪 MSTN基因 焦磷酸测序技术 分子检测 基因分型 |
| DOI：10.11841/j.issn.1007-4333.2019.09.02 |
| 投稿时间：2018-11-20 |
| 基金项目:转基因重大专项（2016ZX08012003）；吉林省科技发展计划项目（20170204012NY）；天津市科普项目（17KPHDSF00040）；农业部农业转基因生物安全评价（食用）重点实验室开放课题 |
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| Pyrosequencing-based detection method for gene editing site |
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LI Congcong1, GAO Yue2,3, SHEN Xiaoling2, LI Feiwu1, ZHAO Xin2, LONG Likun1, LI Liang4, WANG Yong2, LAN Qingkuo2
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| (1.Institute of Agricultural Quality Standard and Testing Technology, Jilin Academy of Agricultural Sciences, Changchun 130033, China;2.Tianjin Institute of Agricultural Quality Standard and Testing Technology, Tianjin 300381, China;3.College of Plant Protection, Hebei Agricultural University, Baoding 071000, China;4.Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China) |
| Abstract: |
| In this study,targeted editing sites of MSTN gene were used as object,and a qualitative and quantitative detection method for gene editing products based on pyrosequencing technology was established.The key parameters of the method included PCR and sequencing primers,dispensation order,standard curve and linearity.The results showed that this method could accurately detect the AG deletion site of exon 3 of MSTN gene and had the functions of qualitative genotyping determination and quantitative detection of allele frequency to accurately determine whether the sample was MSTN gene editing pig product or not and the content of gene editing product.This study would provide technical support for the regulation of gene editing products. |
| Key words: gene editing pig MSTN gene pyrosequencing molecular detection genotyping |
