转基因大豆DBN8205转化体特异性定量PCR方法的研发和验证

doi:10.3864/j.issn.0578-1752.2026.01.003

摘要/Abstract

摘要：

【目的】转基因大豆DBN8205转化体已获批转基因生物安全证书（生产应用），转育品种即将产业化应用，建立DBN8205转化体特异性定性定量检测方法，为转基因生物安全监管和定量标识制度的实施提供依据。【方法】根据DBN8205转化体的分子特征序列，设计转化体特异性引物和探针。通过比较多个引物探针组合的扩增曲线及Ct值，筛选出最佳引物探针组合。分别在实时荧光定量PCR和微滴数字PCR平台上考察DBN8205转化体特异性PCR方法的特异性、检测限、定量限、动力学范围和定量准确性等技术参数。制定标准方法联合验证方案，邀请多家实验室对实时荧光定量PCR方法进行联合验证，对联合验证数据进行统计分析，考察方法的重复性和重现性。【结果】筛选出最佳引物探针组合DBN8205-QF/QR/QP，扩增产物长120 bp，仅特异性识别DBN8205转化体成分，具有良好的扩增特异性。在实时荧光定量PCR平台上，检测限为10 copies，定量限为40 copies，标准曲线的各项技术参数符合标准要求，在40—8.2×10⁴ copies动力学范围内，模板拷贝数和Ct值间具有良好的线性关系，能够对含量低至0.1%的样品进行准确定量。8家实验室联合验证结果表明，DBN8205转化体特异性实时荧光定量PCR方法具有良好的重复性和重现性。在微滴数字PCR平台上，检测限和定量限与实时荧光定量PCR方法相同，分别为10和40 copies，动力学范围为40—8.0×10⁴ copies，能够对低至0.1%的样品进行准确定量，定量结果比qPCR具有更高的精密度。t检验表明，实时荧光定量PCR和二重微滴数字PCR的定量结果具有良好的一致性。【结论】建立的DBN8205转化体特异性定量PCR方法能够对DBN8205转化体进行身份鉴定，可在实时荧光定量PCR和微滴数字PCR平台上对产品中的DBN8205转化体成分进行精准定量分析。

关键词: 转基因大豆, DBN8205转化体, 实时荧光定量PCR, 二重微滴数字PCR, 定量

Abstract:

【Objective】The genetically modified soybean DBN8205 event has been approved for a biosafety certification (for commercial application) in China, and its derived varieties are nearing commercial planting. This study aimed to establish an event-specific detection method for DBN8205 to support the implementation of biosafety regulations and the threshold labeling policy for genetically modified organisms. 【Method】Event-specific primers and probes were designed based on the unique molecular characteristics of the DBN8205 event. The optimal primer/probe set was selected by comparing the amplification curves and Ct values of multiple combinations. The performance of the DBN8205 event-specific PCR method, including specificity, limit of detection (LOD), limit of quantification (LOQ), dynamic range, and quantitative accuracy, was thoroughly evaluated on both real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR) platforms. A collaborative trial was conducted across eight laboratories to validate the qPCR method, with the resulting data statistically analyzed to evaluate its repeatability and reproducibility. 【Result】The primer/probe combination DBN8205-QF/QR/QP was identified as optimal, producing a 120 bp amplicon specifically identified as optimal the DBN8205 event with high specificity. On the qPCR platform, the LOD and LOQ were 10 and 40 copies, respectively. The standard curve exhibited excellent linearity (R²>0.99) over a dynamic range from 40 to 8.2×10⁴ copies, allowing for accurate quantification of samples with DBN8205 content as low as 0.1%. The collaborative validation of eight laboratories confirmed that the qPCR method demonstrated good repeatability and reproducibility. On the ddPCR platform, the LOD and LOQ were identical to those of qPCR. The duplex ddPCR assay also showed a good linear correlation between measured and expected values within the range of 40 to 8.0×10⁴ copies and provided more precise quantification for low-concentration samples (0.1%) compared to qPCR. A t-test indicated no significant difference between the quantitative results obtained from qPCR and ddPCR, demonstrating good consistency between the two platforms. 【Conclusion】The established DBN8205 event-specific PCR method enables unambiguous identification and accurate quantification of the DBN8205 event in products on both qPCR and ddPCR platforms.

Key words: genetically modified soybean, DBN8205 event, real-time quantitative PCR, duplex digital PCR, quantification

吴琼, 谢香庭, 王磊, 牟勇, 李进伟. 转基因大豆DBN8205转化体特异性定量PCR方法的研发和验证[J]. 中国农业科学, 2026, 59(1): 29-40.

WU Qiong, XIE XiangTing, WANG Lei, MOU Yong, LI JinWei. Development and Validation of Event-Specific PCR Method for the Quantification of Genetically Modified Soybean DBN8205[J]. Scientia Agricultura Sinica, 2026, 59(1): 29-40.

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参考文献 29

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靶标 Target	引物/探针 Primer/probe	序列 Sequence (5′-3′)	扩增子大小 Amplicon size (bp)	来源 Reference
DBN8205	DBN8205-QF	TTGAGGGGGTTACGCAATGTT	120	自主研制 Self-developed
	DBN8205-QR	GCGGGGGTCATAACGTGAC
	DBN8205-QP	FAM-TCAGATTGTCGTTTCCCGCCTTCAG-BHQ
lectin	lectin-QF	GCCCTCTACTCCACCCCCA	118	[25]
	lectin-QR	GCCCATCTGCAAGCCTTTTT
	lectin-QP	HEX-AGCTTCGCCGCTTCCTTCAACTTCAC-BHQ

重复 Replication	靶标 Target	曲线斜率 Slope	扩增效率 Amplification efficiency (%)	决定系数 R²
Rep1	DBN8205	-3.245	103.30	0.997
Rep1	lectin	-3.302	100.80	0.997
Rep2	DBN8205	-3.341	99.20	0.998
Rep2	lectin	-3.277	101.90	0.998
Rep3	DBN8205	-3.394	97.10	0.999
Rep3	lectin	-3.445	95.10	0.999

重复 Replicate	S5（100 copies, 0.25%）		S6（40 copies, 0.10%）		S7（20 copies, 0.05%）		S8 (10 copies, 0.025%)
重复 Replicate	DBN8205拷贝数 DBN8205 copies (copies)	百分比 Copy number ratio (%)	DBN8205拷贝数 DBN8205 copies (copies)	百分比 Copy number ratio (%)	DBN8205拷贝数 DBN8205 copies (copies)	百分比 Copy number ratio (%)	DBN8205拷贝数 DBN8205 copies (copies)	百分比 Copy number ratio (%)
1	124	0.309	44	0.123	38	0.102	14	0.037
2	128	0.331	35	0.103	23	0.066	13	0.044
3	104	0.325	40	0.095	29	0.079	10	0.024
4	103	0.288	47	0.117	27	0.079	12	0.039
5	97	0.244	37	0.107	28	0.087	10	0.034
6	99	0.235	40	0.119	13	0.040	6	0.022
7	101	0.274	43	0.128	25	0.079	13	0.040
8	97	0.216	47	0.139	21	0.070	6	0.021
9	115	0.302	40	0.114	21	0.067	16	0.050
10	88	0.242	44	0.126	20	0.060	13	0.038
平均值 Average	106	0.277	42	0.117	25	0.073	11	0.035
相对偏倚 Bias_r (%)	5.60	10.64	4.25	17.10	22.50	46.13	13.00	39.60
标准差 SD (%)	12.74	0.04	4.00	0.01	3.30	0.01	3.30	0.010
相对标准差 RSD (%)	12.06	14.66	9.60	11.08	29.22	27.80	29.22	27.80

样品 Sample	方法 Method	预期含量 Expected content (%)	重复 Replicate			平均值 Average (%)	相对偏倚 Bias_r (%)	标准差 SD (%)	相对标准差 RSD (%)	P值 P value
样品 Sample	方法 Method	预期含量 Expected content (%)	Rep1	Rep2	Rep3	平均值 Average (%)	相对偏倚 Bias_r (%)	标准差 SD (%)	相对标准差 RSD (%)	P值 P value
S1	qPCR	5.0	5.05	5.61	4.95	5.20	4.07	0.39	7.49	0.29
S1	ddPCR	5.0	5.04	4.86	4.93	4.94	-1.15	0.11	2.19	0.29
S2	qPCR	3.0	3.20	2.93	2.91	3.01	0.44	0.17	5.68	0.95
S2	ddPCR	3.0	3.04	2.93	3.05	3.01	0.24	0.07	2.35	0.95
S3	qPCR	1.0	1.03	1.13	1.09	1.08	8.33	0.06	5.45	0.27
S3	ddPCR	1.0	1.06	1.05	1.02	1.04	4.43	0.02	2.31	0.27
S4	qPCR	0.5	0.50	0.55	0.55	0.53	6.67	0.03	5.54	0.19
S4	ddPCR	0.5	0.52	0.49	0.50	0.50	0.85	0.01	2.75	0.19
S6	qPCR	0.10	0.09	0.11	0.10	0.10	0.00	0.01	11.81	0.06
S6	ddPCR	0.10	0.13	0.11	0.13	0.12	24.50	0.01	9.21	0.06

靶标 Target	预期模板量 Expected amount (copy/reaction)	测量值 Measured value (copy/reaction )
靶标 Target	预期模板量 Expected amount (copy/reaction)	Rep1	Rep2	Rep3	均值 Mean	标准差 SD	相对标准差 RSD (%)	相对偏倚 Bias_r (%)
DBN8205	8.0×10⁴	78000	77000	83000	79333	2624.67	3.31	-0.83
	1.0×10⁴	10540	10680	10240	10487	183.55	1.75	4.87
	1.0×10³	1384	1330	1430	1381	40.87	2.96	38.13
	500	506	502	504	504	1.63	0.32	0.80
	50	60	66	60	62	2.83	4.56	24.00
	40	46	40	52	46	4.90	10.65	15.00
Lectin	8.0×10⁴	79600	80000	86400	82000	3115.55	3.80	2.50
	1.0×10⁴	10800	10760	10700	10753	41.10	0.38	7.53
	1.0×10³	1440	1428	1462	1443	14.08	0.98	44.33
	500	488	470	498	485	11.59	2.39	-2.93
	50	56	56	58	57	0.94	1.66	13.33
	40	40	38	42	40	1.63	4.08	0.00
DBN8205/ Lectin	8.0×10⁴	0.98	0.96	0.96	0.97	0.01	0.90	-3.23
	1.0×10⁴	0.98	0.99	0.96	0.98	0.01	1.49	-2.48
	1.0×10³	0.96	0.93	0.98	0.96	0.02	2.02	-4.31
	500	1.04	1.07	1.01	1.04	0.02	2.21	3.90
	50	1.07	1.18	1.03	1.09	0.06	5.58	9.48
	40	1.15	1.05	1.24	1.15	0.08	6.60	14.69