农业用植物基因编辑产品检测方法研究进展

doi:10.3864/j.issn.0578-1752.2024.17.002

摘要/Abstract

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

武玉花, 翟杉杉, 普豪祯, 高鸿飞, 张华, 李俊, 李允静, 肖芳, 吴刚, 徐利群. 农业用植物基因编辑产品检测方法研究进展[J]. 中国农业科学, 2024, 57(17): 3318-3334.

WU YuHua, ZHAI ShanShan, PU HaoZhen, GAO HongFei, ZHANG Hua, LI Jun, LI YunJing, XIAO Fang, WU Gang, XU LiQun. Progress on Detection Methods for Gene-Edited Organisms[J]. Scientia Agricultura Sinica, 2024, 57(17): 3318-3334.

图/表 1

表1

不同基因编辑产品检测方法的优缺点比较"

序号 No.	方法种类 Classification	方法名称 Method	优点 Advantage	缺点 Disadvantage
1	PCR-电泳检测方法 PCR-electrophoresis detection method	PCR-电泳法 PCR-electrophoresis method	只需常规设备，操作门槛低，适用于编辑产品的筛查 Requires only routine equipment, a low operational threshold, and suitable for the screening of edited products	操作步骤繁琐，PCR反应后，还需要电泳等操作；受限于电泳的分辨率，不能检测单碱基或相同大小片段的替换；不能进行特异基因编辑产品的识别检测 The procedure is quite cumbersome, requiring additional steps after PCR, such as electrophoresis; it is limited by the resolution of the electrophoresis, which cannot detect single base substitutions or replacements of identically sized fragments; and it cannot perform specific identification of gene- edited products
1	PCR-电泳检测方法 PCR-electrophoresis detection method	PCR-酶切-电泳法 PCR-digestion- electrophoresis method		操作步骤繁琐；依赖酶切位点的有无；不能进行特异编辑产品的识别检测 The operation steps are cumbersome; it relies on the presence or absence of enzyme sites; it cannot perform specific identification of GE products
2	基于测序的检测方法 Sequencing-based detection method	Sanger测序法 Sanger sequencing method	通用性强，可检测一切变异 Universal, capable of detecting all types of variations	通常依赖第三方测序公司，耗时 Relies on third-party sequencing companies, time- consuming
		高通量测序法 High-throughput sequencing method	适用范围广，可检测脱靶及载体骨架残留 A broad range of applications, capable of detecting off-target as well as residual vector backbone	通常依赖第三方测序公司，耗时；数据分析复杂 Relies on third-party sequencing companies, time- consuming, and the data analysis is complex
		焦磷酸测序法 Pyrophosphate sequencing method	准确性高，速度快 High accuracy and fast speed	通常依赖第三方测序公司，耗时；检测瞬时发光，通量受限；只能测短序列 Relies on third-party sequencing companies, time- consuming, detecting transient luminescence, the throughput is limited and only capable of measuring short sequences
3	基于实时荧光PCR的检测方法 Real-time PCR-based detection method	TaqMan探针法 TaqMan probe method	特异性强、灵敏度高、速度快、不易交错污染；设计不同的TaqMan探针，可进行编辑产品的筛查和特异识别；可定量检测 Strong specificity, high sensitivity, rapid speed, and low risk of cross-contamination; it allows for the screening and specific identification of GE products by designing different TaqMan probes; and it enables quantitative detection	1—2个碱基的变异，难以设计满足特异性要求的TaqMan探针 Variations of 1-2 bp make it difficult to design TaqMan probes that meet the specificity requirements
		高分辨率熔解曲线法 High-resolution melting analysis	采用荧光染料，成本低；分析荧光曲线，速度快；可进行编辑产品的筛查 Using fluorescent dyes, low cost; analyzing the fluorescence curve is fast; allows for the screening of GE products	不能识别熔解曲线相似的不同产品；只能筛查样品是否被编辑，不能对特定的基因编辑产品进行鉴定 It cannot distinguish between different products with similar melting curves; it can only screen whether the sample has been edited, but cannot identify specific GE products
		KASP方法 KASP method	采用通用性探针，成本低；检测荧光信号，速度快；适合单碱基变异基因编辑产品的检测；对野生型和编辑型同步检测 Using universal probes, low cost; rapid speed; suitable for the detection of GE products with SNV; and it allows for simultaneous detection of both wild-type and edited type	引物设计位点受限，部分序列难以设计特异性引物；只有一种野生型或编辑型模板时，有非特异性扩增信号，与低含量产品难区分 Primer design sites are limited, and it is difficult to design specific primers for some sequences; when only wild-type or edited template is present in PCR, there can be non-specific amplification, making it difficult to distinguish from low-level products
		ARMs-qPCR法 ARMs-qPCR method	特异性提高，能识别单碱基变异基因编辑产品 Specificity is enhanced, allowing the identification of GE products with SNVs	引物设计位点受限，部分序列难以设计引物；引入错配碱基，导致方法的灵敏度下降 Primer design is site-limited, making it difficult to design primers for some sequences; the introduction of mismatched bases can lead to a decrease in the sensitivity of the method
		rhPCR法 rhPCR method	特异性强，灵敏度高，速度快，能识别单碱基变异基因编辑产品 Strong specificity, high sensitivity, fast speed, capable of identifying GE products with SNVs	引物成本较高；对发生单碱基缺失的基因编辑产品，难以设计引物 Primer costs are relatively high; it is difficult to design primers for GE products with a single base deletion
4	基于数字PCR的检测方法 Digital PCR-based detection method	TaqMan探针法 TaqMan probe method	与基于实时荧光PCR的TaqMan探针法兼容；适用于基因编辑产品的精准定量检测；适用于评价动物细胞的编辑效率 Compatible with the TaqMan probe method based on qPCR; suitable for precise quantitative detection of GE products; suitable for evaluating the editing efficiency of animal cells	当前成本高；1—2个碱基的变异，难以设计满足特异性要求的TaqMan探针 Current costs are high; variations of 1-2 base pairs make it difficult to design TaqMan probes that meet the specificity requirements
5	基于可编程内切酶的检测方法 Cas-based detection method	基于Cas9的检测技术 Cas9-based method	能识别单碱基变异基因编辑产品 Capable of identifying GE products with SNVs	受限于突变位点附近PAM位点的有无；酶切产物需要进一步分析，操作步骤比较繁琐 Limited by the presence or absence of PAM sites near the mutation site; the digestion products require further analysis, making the operational steps more cumbersome
5	基于可编程内切酶的检测方法 Cas-based detection method	基于Cas12、Cas13、Cas14的检测技术 Cas12, Cas13 and Cas14-based method	能识别单碱基变异基因编辑产品；通过反式切割信号分子发出信号，适合与核酸预扩增集成，适用于现场检测 Capable of identifying GE products with SNVs; it emits fluorescent signal by a trans-cleavage of signaling molecule, suitable for on-site testing by integrating with pre-amplification of DNA	受限于突变位点附近PAM位点的有无 Limited by the presence or absence of PAM sites near the mutation site
6	基于仪器的检测方法 Instrument-based detection method	基因芯片法、质谱法等 Gene chip method, mass spectrometry, etc	自动化分析，通量高，速度快 Automated analysis, high throughput, and fast speed	设备昂贵，成本高昂 The equipment is expensive, resulting in high costs

表1

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