Development of Multi-target Plasmid DNA Reference Materials for Screening Transgenic Soybeans

doi:10.19586/j.2095-2341.2026.0029

Current Biotechnology ›› 2026, Vol. 16 ›› Issue (4): 863-877.DOI: 10.19586/j.2095-2341.2026.0029

• Special Forum on Detection Technology for Genetically Modified Organisms • Previous Articles Next Articles

Development of Multi-target Plasmid DNA Reference Materials for Screening Transgenic Soybeans

Pengfei LIU¹(), Na LIU¹, Ruihuan LI¹, Wei FU², Chao XU¹, Qingkuo LAN¹, Yong WANG¹, Xin ZHAO¹()

^1.Institute of Germplasm Resources and Biotechnologe，Tianjin Academy of Agricultural Sciences，Tianjin 300384，China
^2.Development Center of Science and Technology，Ministry of Agriculture and Rural Affairs，Beijing 100176，China

Received:2026-02-09 Accepted:2026-03-13 Online:2026-07-25 Published:2026-09-11
Contact: Xin ZHAO

Abstract

Abstract:

Soybeans are rich in plant proteins and edible oils， and also provide beneficial nutrients such as isoflavones and lecithin， making them important crops for food， feed， and oil production. China's demand for and import volume of soybeans have increased year by year， with import dependence at times exceeding 80%. Five transgenic soybean events （DBN9004， DBN8002， SHZD32-1， ZH6106， and CAL16）， which were independently developed in China and have obtained biosafety certificates for genetically modified organisms， were used as research objects. On the basis of the standard qualitative and quantitative detection methods for each transgenic event and its derivative varieties， the five transgenic soybean events and the endogenous standard gene Lectin were assembled into a binary vector. A series of experiments， including plasmid molecular verification， screening of plasmid preservation medium， method specificity validation， and detection applicability and interchangeability testing using genomic DNA， was then carried out to develop the plasmid DNA reference molecule pUC-soy-identification for transgenic screening. The results showed that the sequence of the prepared plasmid reference molecule was completely consistent with the target sequence， and both restriction enzyme digestion and qPCR amplification produced the expected results. The addition of salmon sperm DNA helped bring the copy number ratio closer to the theoretical value. The plasmid preparation met the specified criteria for use as a positive control in specificity， applicability， and interchangeability tests targeting DBN9004， DBN8002， SHZD32-1， ZH6106， and CAL16. Therefore， the resulting plasmid reference molecule can be applied to the quantitative detection of the aforementioned transgenic soybeans and their processed products， and provides a robust technical tool for the biosafety management and labeling compliance of genetically modified organisms in China.

Key words: transgenic soybean, plasmid reference materials, applicability, specificity

CLC Number:

Q-33

Pengfei LIU, Na LIU, Ruihuan LI, Wei FU, Chao XU, Qingkuo LAN, Yong WANG, Xin ZHAO. Development of Multi-target Plasmid DNA Reference Materials for Screening Transgenic Soybeans[J]. Current Biotechnology, 2026, 16(4): 863-877.

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URL: https://swjsjz.magtechjournal.com/EN/10.19586/j.2095-2341.2026.0029

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Figures/Tables 18

References 21

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靶标	引物序列（5′→3′）	产物大小/bp	来源
DBN9004	F：5′-TGCTTCCAATTTGCCACACGCTAGCAGTAACATTGATC-3′	433	自行设计
DBN9004	R：5′-GTTTAAACTGAAGGCGGGAAACGACAATCTGATCCAAGCTCAAGC-3′	433	自行设计
DBN8002	F：5′-TGGTTGATGGAGAGGAGGGTGGCGCCTGGCCGTATCCGC-3′	267	自行设计
DBN8002	R：5′-GTTAAACAATAGAAATTAAAAAAACCTAACAACAAATGTCTTGATTTTAAAGAC-3′	267	自行设计
SHZD32-1	F：5′-CAACAGTTGCGCAGCCTGAATGGCGAATGCTAGAGCAGC-3′	208	自行设计
SHZD32-1	R：5′-GCGGATACGGCCAGGCGCCACCCTCCTCTCCATCAACC-3′	208	自行设计
中黄6106	F：5′-GCTTGAGCTTGGATCAGATTGTCGTTTCCCGCCTTCAGTTTAAAC-3′	250	自行设计
中黄6106	R：5′-GCTGCTCTAGCATTCGCCATTCAGGCTGCGCAACTGTTG-3′	250	自行设计
CAL16	F：5′-GTCTTTAAAATCAAGACATTTGTTGTTAGGTTTTTTTAATTTCTATTGTTTAAC-3′	213	自行设计
CAL16	R：5′-CAAGAGTACATTAAAAACGTCCGCAATGTGTTATTAAGTTGTCCAC-3′	213	自行设计
Lectin	F：5′-GTGGACAACTTAATAACACATTGCGGACGTTTTTAATGTACTCTTG-3′	118	自行设计
Lectin	R：5′-AGGAAACAGCTATGACCATGCTCGAGCCAAGCTCGGCGCG-3′	118	自行设计

靶标	引物序列（5′→3′）	产物大小/bp	来源
DBN9004	F：5′-TGCTTCCAATTTGCCACACGCTAGCAGTAACATTGATC-3′	433	自行设计
DBN9004	R：5′-GTTTAAACTGAAGGCGGGAAACGACAATCTGATCCAAGCTCAAGC-3′	433	自行设计
DBN8002	F：5′-TGGTTGATGGAGAGGAGGGTGGCGCCTGGCCGTATCCGC-3′	267	自行设计
DBN8002	R：5′-GTTAAACAATAGAAATTAAAAAAACCTAACAACAAATGTCTTGATTTTAAAGAC-3′	267	自行设计
SHZD32-1	F：5′-CAACAGTTGCGCAGCCTGAATGGCGAATGCTAGAGCAGC-3′	208	自行设计
SHZD32-1	R：5′-GCGGATACGGCCAGGCGCCACCCTCCTCTCCATCAACC-3′	208	自行设计
中黄6106	F：5′-GCTTGAGCTTGGATCAGATTGTCGTTTCCCGCCTTCAGTTTAAAC-3′	250	自行设计
中黄6106	R：5′-GCTGCTCTAGCATTCGCCATTCAGGCTGCGCAACTGTTG-3′	250	自行设计
CAL16	F：5′-GTCTTTAAAATCAAGACATTTGTTGTTAGGTTTTTTTAATTTCTATTGTTTAAC-3′	213	自行设计
CAL16	R：5′-CAAGAGTACATTAAAAACGTCCGCAATGTGTTATTAAGTTGTCCAC-3′	213	自行设计
Lectin	F：5′-GTGGACAACTTAATAACACATTGCGGACGTTTTTAATGTACTCTTG-3′	118	自行设计
Lectin	R：5′-AGGAAACAGCTATGACCATGCTCGAGCCAAGCTCGGCGCG-3′	118	自行设计

靶标	引物/探针序列（5′→3′）	产物大小/bp	来源
DBN9004	F：5′-AACGCGGCCGCTCTAG-3′	165	待发布国家标准
	R：5′-ATTATGTGGAAAGAAATGACCGAAA-3′
	P：5′-CGCCGGGTCCCGTTTAAACTATCAGT-3′
DBN8002	F：5′-TATCCGCAATGTGTTATTAAGTTGTC-3′	110	待发布国家标准
	R：5′-ATGGGACACTTGAGGAATGACA-3′
	P：5′-TGTGTAACGCTTGTTTTGCTAT-3′
SHZD32-1	F：5′-TCGTTTCCCGCCATAAGG-3′	120	农业农村部864号公告—15—2024
	R：5′-CATCAACCAAGAGCAACAGCAT-3′
	P：5′-FAM-TCCGACCACCACGAGACCGTAGTACA-BHQ1-3′
中黄6106	F：5′-TCTAAGCGTCAATTTGTTTACATCA-3′	96	农业农村部864号公告—16—2024
	R：5′-AAAGGTGTGAAATGTGAACGC-3′
	P：5′-FAM-TTACCTAAAACATCTCAGCACATCGC-BHQ1-3′
CAL16	F：5′-GTTGCTACATGTTGACTGATTATTTCTTT-3′	112	待发布国家标准
	R：5′-CGCAATGTGTTATTAAGTTGTCCACTA-3′
	P：5′-FAM- CTCATTGATAAAAGATAAGCC -MGB-3′
Lectin	F：5′-GCCCTCTACTCCACCCCCA-3′	118	农业部2031号公告—8—2013
	R：5′-GCCCATCTGCAAGCCTTTTT-3′
	P：5′-HEX-AGCTTCGCCGCTTCCTTCAACTTCAC-BHQ1-3′

靶标	引物/探针序列（5′→3′）	产物大小/bp	来源
DBN9004	F：5′-AACGCGGCCGCTCTAG-3′	165	待发布国家标准
	R：5′-ATTATGTGGAAAGAAATGACCGAAA-3′
	P：5′-CGCCGGGTCCCGTTTAAACTATCAGT-3′
DBN8002	F：5′-TATCCGCAATGTGTTATTAAGTTGTC-3′	110	待发布国家标准
	R：5′-ATGGGACACTTGAGGAATGACA-3′
	P：5′-TGTGTAACGCTTGTTTTGCTAT-3′
SHZD32-1	F：5′-TCGTTTCCCGCCATAAGG-3′	120	农业农村部864号公告—15—2024
	R：5′-CATCAACCAAGAGCAACAGCAT-3′
	P：5′-FAM-TCCGACCACCACGAGACCGTAGTACA-BHQ1-3′
中黄6106	F：5′-TCTAAGCGTCAATTTGTTTACATCA-3′	96	农业农村部864号公告—16—2024
	R：5′-AAAGGTGTGAAATGTGAACGC-3′
	P：5′-FAM-TTACCTAAAACATCTCAGCACATCGC-BHQ1-3′
CAL16	F：5′-GTTGCTACATGTTGACTGATTATTTCTTT-3′	112	待发布国家标准
	R：5′-CGCAATGTGTTATTAAGTTGTCCACTA-3′
	P：5′-FAM- CTCATTGATAAAAGATAAGCC -MGB-3′
Lectin	F：5′-GCCCTCTACTCCACCCCCA-3′	118	农业部2031号公告—8—2013
	R：5′-GCCCATCTGCAAGCCTTTTT-3′
	P：5′-HEX-AGCTTCGCCGCTTCCTTCAACTTCAC-BHQ1-3′

	1次	2次	3次	4次	5次	6次	平均值	SD	RSD
OD₂₆₀/OD₂₈₀	1.81	1.86	1.82	1.84	1.88	1.82	1.84	0.03	0.01
OD₂₆₀/OD₂₃₀	2.10	2.03	2.07	2.09	2.11	2.02	2.07	0.04	0.02
QuantiFluor染料法浓度/（ng·μL^-1）	133.10	132.70	133.90	136.80	135.60	134.80	134.48	1.56	0.01

Development of Multi-target Plasmid DNA Reference Materials for Screening Transgenic Soybeans

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靶标	保存介质	冻融次数	酵母tRNA	鲑鱼精DNA	0.1×TE
DBN9004	转化体特异性序列拷贝数/(copies·μL^-1)	0次	5 900	10 677	1 600
	内标拷贝数/(copies·μL^-1)		5 737	10 600	1 733
	DBN9004转化体Lectin拷贝数比值/%		1.03	1.01	0.93
	转化体特异性序列拷贝数/(copies·μL^-1)	10次	5 233	10 233	1 700
	内标拷贝数/(copies·μL^-1)		6 000	10 233	1 700
	DBN9004转化体Lectin拷贝数比值/%		0.87	1.00	1.01
DBN8002	转化体特异性序列拷贝数/(copies·μL^-1)	0次	7 067	11 833	2 333
	内标拷贝数/(copies·μL^-1)		7 233	11 833	2 467
	DBN8002转化体Lectin拷贝数比值/%		0.98	1.00	0.96
	转化体特异性序列拷贝数/(copies·μL^-1)	10次	6 300	11 600	2 000
	内标拷贝数/(copies·μL^-1)		6 867	10 767	2 033
	DBN8002转化体Lectin拷贝数比值/%		0.92	0.98	1.00
CAL16	转化体特异性序列拷贝数/(copies·μL^-1)	0次	5 900	10 933	1 567
	内标拷贝数/(copies·μL^-1)		6 133	10 600	1 700
	CAL16转化体Lectin拷贝数比值/%		0.96	1.03	0.94
	转化体特异性序列拷贝数/(copies·μL^-1)	10次	5 433	10 300	1 500
	内标拷贝数/(copies·μL^-1)		6 133	10 300	2 033
	CAL16转化体Lectin拷贝数比值/%		0.89	1.00	0.75
SHZD32-1	转化体特异性序列拷贝数/(copies·μL^-1)	0次	6 100	10 567	1 900
	内标拷贝数/(copies·μL^-1)		6 167	10 500	1 533
	SHZD32-1转化体Lectin拷贝数比值/%		0.99	1.01	1.30
	转化体特异性序列拷贝数/(copies·μL^-1)	10次	5 600	9 967	1 967
	内标拷贝数/(copies·μL^-1)		6 300	10 167	2 067
	SHZD32-1转化体Lectin拷贝数比值/%		0.89	0.98	0.98
中黄6106	转化体特异性序列拷贝数/(copies·μL^-1)	0次	6 867	11 267	1 767
	内标拷贝数/(copies·μL^-1)		6 733	10 933	1 933
	中黄6106转化体Lectin拷贝数比值/%		1.02	1.03	0.92
	转化体特异性序列拷贝数/(copies·μL^-1)	10次	6 300	10 200	1 600
	内标拷贝数/(copies·μL^-1)		6 677	10 467	2 300
	中黄6106转化体Lectin拷贝数比值/%		0.95	0.97	0.70

		Lectin	DBN9004	DBN8002	中黄6106	SHZD32-1	CAL16
决定系数（R²）	1	0.999	0.996	0.998	0.999	0.999	0.996
	2	0.995	0.998	0.998	0.998	0.999	0.999
	3	0.996	0.999	0.991	0.994	0.998	0.994
	平均值	0.997	0.998	0.996	0.997	0.999	0.996
	RSD	0.21	0.15	0.41	0.27	0.06	0.25
斜率	1	-3.329	-3.152	-3.456	-3.143	-3.233	-3.559
	2	-3.340	-3.163	-3.462	-3.185	-3.197	-3.384
	3	-3.401	-3.197	-3.392	-3.192	-3.250	-3.403
	平均值	-3.357	-3.171	-3.437	-3.173	-3.227	-3.449
	RSD	-1.16	-0.74	-1.13	-0.84	-0.84	-2.78
PCR扩增效率	1	99.7	101.6	98.7	100.5	100.8	95.6
	2	99.4	100.1	100.6	98.3	97.5	97.5
	3	96.9	98.1	97.1	100.9	98.1	96.7
	平均值	98.68	99.93	98.80	99.90	98.80	96.60
	RSD	1.54	1.76	1.77	1.40	1.78	0.99

质粒DNA各靶标标准曲线参数		基因组DNA各靶标标准曲线
质粒DNA各靶标标准曲线参数		Lectin	DBN9004	DBN8002	中黄6106	SHZD32-1	CAL16
R²	Lectin	0.538
	DBN9004		0.101
	DBN8002			0.451
	中黄6106				0.882
	SHZD32-1					0.051
	CAL16						0.214
斜率	Lectin	0.125
	DBN9004		0.861
	DBN8002			0.055
	中黄6106				0.215
	SHZD32-1					0.373
	CAL16						0.547
扩增效率（E）	Lectin	0.756
	DBN9004		0.696
	DBN8002			0.504
	中黄6106				0.589
	SHZD32-1					0.307
	CAL16						0.387

[1]	Feng ZHANG, Xiaoyun HE, Kunlun HUANG, Qihuai WANG. Food Safety Evaluation of Transgenic Herbicide Tolerant Soybean [J]. Current Biotechnology, 2021, 11(4): 489-495.
[2]	MIAO Qingmei1, ZHAO Yang2, XU Xiaoli1, XU Junfeng1, WANG Xiaofu1, CHEN Xiaoyun1*. Establishment of Transformant-specific Detection Method for the Herbicide-tolerant Transgenic Soybean Event ZUTS-33 Harboring the g10-epsps Transgene [J]. Curr. Biotech., 2020, 10(6): 696-703.

靶标		Lectin	DBN9004	DBN8002	中黄6106	SHZD32-1	CAL16
决定系数（R²）	1	0.999	0.994	0.993	0.999	0.996	0.992
	2	0.996	0.996	0.991	0.997	0.993	0.995
	3	0.998	0.996	0.996	0.994	0.995	0.994
	平均值	0.998	0.995	0.993	0.997	0.995	0.994
	RSD	0.150	0.120	0.250	0.250	0.150	0.150
斜率	1	-3.321	-3.193	-3.345	-3.061	-3.125	-3.436
	2	-3.313	-3.151	-3.286	-3.180	-3.179	-3.395
	3	-3.330	-3.178	-3.387	-3.095	-3.250	-3.400
	平均值	-3.321	-3.174	-3.339	-3.112	-3.185	-3.410
	RSD	-0.260	-0.670	-1.520	-1.970	-1.970	-0.660
PCR扩增效率	1	99.40	98.90	96.90	100.90	99.80	96.80
	2	99.10	100.20	99.60	97.50	99.400	97.10
	3	96.20	99.30	97.10	99.10	101.3	97.80
	平均值	98.23	99.47	97.87	99.17	100.17	97.23
	RSD	1.80	0.67	1.54	1.72	1.00	0.53

靶标		Lectin	DBN9004	DBN8002	中黄6106	SHZD32-1	CAL16
决定系数（R²）	1	0.999	0.994	0.993	0.999	0.996	0.992
	2	0.996	0.996	0.991	0.997	0.993	0.995
	3	0.998	0.996	0.996	0.994	0.995	0.994
	平均值	0.998	0.995	0.993	0.997	0.995	0.994
	RSD	0.150	0.120	0.250	0.250	0.150	0.150
斜率	1	-3.321	-3.193	-3.345	-3.061	-3.125	-3.436
	2	-3.313	-3.151	-3.286	-3.180	-3.179	-3.395
	3	-3.330	-3.178	-3.387	-3.095	-3.250	-3.400
	平均值	-3.321	-3.174	-3.339	-3.112	-3.185	-3.410
	RSD	-0.260	-0.670	-1.520	-1.970	-1.970	-0.660
PCR扩增效率	1	99.40	98.90	96.90	100.90	99.80	96.80
	2	99.10	100.20	99.60	97.50	99.400	97.10
	3	96.20	99.30	97.10	99.10	101.3	97.80
	平均值	98.23	99.47	97.87	99.17	100.17	97.23
	RSD	1.80	0.67	1.54	1.72	1.00	0.53