CRISPR/Cas9基因编辑高GABA番茄快速检测方法研究

doi:10.16035/j.issn.1001-7283.2026.05.019

摘要/Abstract

摘要：

针对CRISPR/Cas9基因编辑的高γ-氨基丁酸（GABA）番茄建立了荧光PCR和数字PCR检测方法，通过高GABA番茄与野生型番茄基因序列对比，设计多组特异性引物探针序列，利用荧光PCR法检测得出1组符合要求的引物探针序列，检测灵敏度可达0.01%，稳定性良好，且在数字PCR检测体系中同样具有特异性。荧光PCR检测耗时短，适合常规定性检测，成本较低，但灵敏度和精准度有限；数字PCR操作复杂、成本高且检测耗时较长，但无需标准曲线，绝对定量更精准，尤其适合低丰度样本或复杂背景下的微量检测。该方法的建立为基因编辑植物的检测提供了方法依据，并为制订相关检测标准打下基础，为生物安全监管提供技术支撑。

关键词: 番茄, 基因编辑, γ-氨基丁酸, 荧光PCR, 数字PCR

Abstract:

Our laboratory established fluorescence PCR and digital PCR detection methods for the high gamma- aminobutyric acid (GABA) tomato edited by CRISPR/Cas9. By comparing the gene sequences of high-GABA tomato and wild-type tomato, we designed multiple groups of specific primer-probe sequences, and detected a set of primer probe sequences that met the detection requirements by fluorescent PCR. The detection sensitivity can reach 0.01%, the stability was good, and it was also specific in the digital PCR detection system. Fluorescent PCR detection took a short time and was suitable for routine qualitative detection, with low cost, but its sensitivity and accuracy are limited. Digital PCR was complex in operation, high in cost and long in detection time, but it did not need a standard curve, and its absolute quantification was more accurate, especially suitable for trace detection in low abundance samples or complex background. The establishment of this method provides a methodological basis for the detection of genetically edited plants, lays a foundation for formulating relevant detection standards, and provides technical support for biosafety supervision.

Key words: Tomato, Gene editing, Gamma-aminobutyric acid, Fluorescent PCR, Digital PCR

袁小雅, 甄珍. CRISPR/Cas9基因编辑高GABA番茄快速检测方法研究[J]. 作物杂志, 2026 (5): 166–170

Yuan Xiaoya, Zhen Zhen. Rapid Detection Method for High-GABA Tomato Edited by CRISPR/Cas9[J]. Crops, 2026(5): 166–170

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名称 Name		序列 Sequence (5′→3′)	扩增大小 Amplification size (bp)
G1	G1-F	GGAGCACATGTACAATGCTTTACC	99
	G1-R	CGGCAATGTGTTTAATTGCTTAG
	G1-P	GAAACAACCCGCCTGGTTGAT
G2	G2-F	GCACATGTACAATGCTTTACC	98
	G2-R	GTGTTTAATTGCTTAGTGGCAGC
	G2-P	GAACGAGACTGCCTGGTTGATGC
G3	G3-F	GCACATGTACAATGCTTTACCAAC	99
	G3-R	GCAATGTGTTTAATTGCTTAGTGGC
	G3-P	GGAAACAACCCGCCTGGTT
G4	G4-F	GCACATGTACAATGCTTTACC	97
	G4-R	GTGTTTAATTGCTTAGTGGCAGC
	G4-P	GAACGAGACTGCCTGGTTGATGC

浓度 Concentration (%)	C_t			平均C_t Average C_t	相对标准偏差 RSD (%)
浓度 Concentration (%)	1	2	3	平均C_t Average C_t	相对标准偏差 RSD (%)
5.00	30.89	32.12	31.13	31.38	2.08
1.00	31.96	32.78	31.86	32.20	1.57
0.50	32.86	33.68	32.82	33.12	1.46
0.10	34.12	35.08	35.44	34.88	1.97
0.05	35.90	36.76	36.27	36.31	1.19
0.01	38.11	38.88	38.60	38.53	1.01
0.005	ND	39.62	ND	ND	ND

序号Number	国家Country	品种名称Variety name
1	中国	诺盾314
2	中国	荷兰德奥特302
3	中国	德澳特7845
4	日本	心形番茄
5	日本	阳光大道
6	德国	鲁比阿姨的德国绿番茄
7	德国	巴斯夫蜜思提拉小番茄
8	荷兰	德奥特302
9	俄罗斯	黑柿子番茄
10	韩国	釜山金粉
11	法国	樱桃番茄
12	美国	传统牛排番茄
13	美国	紫色番茄