中国农业科学 ›› 2024, Vol. 57 ›› Issue (17): 3318-3334.doi: 10.3864/j.issn.0578-1752.2024.17.002

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

农业用植物基因编辑产品检测方法研究进展

武玉花1(), 翟杉杉1, 普豪祯1,2, 高鸿飞1, 张华3, 李俊1, 李允静1, 肖芳1, 吴刚1, 徐利群3()   

  1. 1 中国农业科学院油料作物研究所/农业农村部农业转基因生物溯源重点实验室,武汉 430062
    2 中南民族大学,武汉 430074
    3 农业农村部科技发展中心,北京 100025
  • 收稿日期:2024-02-26 接受日期:2024-04-26 出版日期:2024-09-01 发布日期:2024-09-04
  • 通信作者:
    武玉花,E-mail:
    徐利群,E-mail:
  • 基金资助:
    科技创新2030—重大项目(2022ZD0402010)

Progress on Detection Methods for Gene-Edited Organisms

WU YuHua1(), ZHAI ShanShan1, PU HaoZhen1,2, GAO HongFei1, ZHANG Hua3, LI Jun1, LI YunJing1, XIAO Fang1, WU Gang1, XU LiQun3()   

  1. 1 Oil Crops Research Institute, Chinese Academy of Agricultural Sciences/Key Laboratory of Agricultural Genetically Modified Organism Traceability, Ministry of Agriculture and Rural Affairs, Wuhan 430062
    2 South-Central Minzu University, Wuhan 430074
    3 Development Center of Science and Technology, Ministry of Agriculture and Rural Affairs, Beijing 100025
  • Received:2024-02-26 Accepted:2024-04-26 Published:2024-09-01 Online:2024-09-04

摘要:

随着基因编辑技术的兴起,基因编辑产品已经从实验室走向产业化应用,2022年农业农村部针对没有引入外源基因的基因编辑植物的安全评价,专门出台了《农业用基因编辑植物安全评价指南(试行)》,2023年为基因编辑大豆AE15-18-1颁发了首个生物安全证书,标志着我国正式开启了基因编辑作物的产业化进程。基因编辑产品不同于传统转基因产品,不含有外源DNA序列,常用的转基因检测策略不适用于基因编辑产品的检测。随着农作物基因编辑产品产业化进程的积极推进,如何高效准确检测是否为基因编辑产品及其编辑特征,是关系到基因编辑产品产业化利用和知识产权保护的重要依据,急需开发适用于基因编辑产品的检测技术。以检测靶标序列是否被编辑为目标,基于PCR、测序等技术开发出了很多检测技术,并广泛应用于产品研发过程中基因编辑产品的筛选。产业化后的安全监管和知识产权保护,不仅要检测样品是否被编辑,还要快速识别待测样品的核苷酸序列特征,确定样品的来源和身份,进而对基因编辑成分进行精准定量,判定是否需要定量标识。目前,对于含有少数几个碱基插入、缺失及单碱基变异等突变的基因编辑产品,难以应用常规PCR或测序等技术进行快速的身份鉴定,更难以对基因编辑成分的含量进行精准定量检测。以快速识别编辑后的DNA序列特征、并精准定量为目标,根据基因编辑产品的分子特征,本文综述了基于凝胶电泳的普通PCR法、基于测序的检测方法、基于实时荧光PCR的检测方法、基于数字PCR的检测方法、基于可编程内切酶的方法,以及基于仪器的检测方法在基因编辑产品检测中的应用及优缺点,以期探索出适用于基因编辑产品的检测和定量策略,为后续基因编辑产品检测方法的开发提供参考。

关键词: 基因编辑, 测序, 实时荧光PCR, 数字PCR, 可编程内切酶

Abstract:

Gene editing techniques have made gene edited (GE) organisms enter commercial applications from laboratories. In 2022, the Ministry of Agriculture and Rural Affairs specifically issued the “Guidelines for Safety Evaluation of Genetically Edited Plants for Agricultural Use (Trial)” for the safety evaluation of GE plants without introducing exogenous genes. In 2023, China granted the first biosafety certificate for GE soybean AE15-18-1, marking the official start of the commercialization process of GE crops in China. GE organisms are different from traditional genetically modified organisms (GMOs) containing exogenous DNA sequences, making common GM detection strategies inapplicable to the detection of GE organisms. As the industrialization of GE crops progresses positively, how to efficiently and accurately detect whether a product is gene-edited and its editing characteristics is an important basis for the commercial use and intellectual property protection of GE products. There is an urgent need to develop detection technologies suitable for GE products. With the goal of detecting whether the target sequence has been edited, many detection technologies have been developed based on PCR, sequencing, and other technologies, and are widely used in the screening of GE products in the research and development process. After industrialization, safety supervision and intellectual property protection require not only the detection of whether the sample has been edited but also the rapid identification of the nucleotide sequence characteristics of the sample to determine its origin and identity. Subsequently, precise quantification of the GE components is necessary to determine whether quantitative labeling is required. Currently, it is difficult to quickly identify the identity of GE products with only a few base insertions, deletions, and single nucleotide variations (SNV) using conventional PCR or sequencing technologies. It is even more challenging to accurately quantify the content of GE components. Aiming at the rapid identification of the DNA sequence characteristics after editing and precise quantification, based on the molecular characteristics of GE products, this paper reviews the application of the gel electrophoresis-based PCR method, the sequencing-based method, the real-time PCR-based method, the digital PCR-based method, the editing enzyme-based method, and the instrument-based method in detection of GE organisms, and expounds the advantages and disadvantages of each method during detection. This review initially explores the detection and quantification strategies suitable for GE organisms and provides a reference for subsequent development of detection methods for GE organisms.

Key words: gene editing, sequencing, real time PCR, digital PCR, editing enzyme