转基因大豆WYN341GmC数字PCR精准定量检测方法的建立

doi:10.3864/j.issn.0578-1752.2026.14.003

摘要/Abstract

摘要：

【目的】基于微滴式数字PCR（ddPCR）技术，建立一种特异性强、灵敏度高的定量检测方法，为转基因大豆WYN341GmC的精准定量检测及安全监管提供可靠技术支撑。【方法】以转基因大豆WYN341GmC转化体边界序列为靶标，设计特异性引物与探针，优化反应体系参数（包括引物探针浓度），并通过特异性、灵敏度、线性范围、正确度和精密度等指标验证方法性能。在方法筛选阶段主要看以下5点：热图中双通道微滴信号稳定、阳性/阴性微滴能分开、雨滴数量少、有效微滴数量充足，以及WYN341GmC和Lectin的拷贝数比值接近理论值。在方法验证阶段主要采用ddPCR技术，结合标准品梯度稀释试验，对方法的定量限（LOQ）、检出限（LOD）、正确度和精密度进行评估。【结果】以WYN341GmC转化体特异性序列和大豆内标基因Lectin为检测对象，建立了二重ddPCR精准定量方法。综合上述方法中的各项筛选指标，最终确定采用特异性引物探针组合WYN341LB-QF2/LB-QR1/LB-QP1与内标引物探针组合LectinQF/QR/QP进行后续试验。体系参数方面，两套引物/探针选择相同的浓度配置，引物浓度为0.8 μmol·L^-1、探针浓度为0.4 μmol·L^-1，退火延伸温度最终确定为58 ℃，在上述二重ddPCR方法条件下，转化体与内标微滴聚类清晰、分群稳定，结果精密度较好。性能验证结果表明，该方法的检出限为8 copies，定量限为17 copies，可满足可靠定量；在17—2.08×10⁴copies动力学范围内，WYN341GmC转化体的回归曲线决定系数R²为1.000，Lectin的回归曲线决定系数R²为0.999，证明理论模板拷贝数与实际模板拷贝数之间存在良好的线性关系，适合在动力学范围内进行定量检测。将该方法用于大豆盲样测定时，能够对WYN341GmC含量进行准确定量，并且保证定值的偏差在25%以下。【结论】建立了转基因大豆WYN341GmC二重ddPCR定量方法，明确了检测靶标、反应体系及条件，并完成了相关方法学验证，可使用该方法在ddPCR平台上对WYN341GmC转化体含量进行精准定量检测。

关键词: 二重微滴式数字PCR, 转基因大豆WYN341GmC, 精准定量, 转化体特异性, 方法学验证

Abstract:

【Objective】To establish a quantitative detection method with high specificity and sensitivity based on droplet digital PCR (ddPCR), thereby providing reliable technical support for the precise quantification and safety supervision of genetically modified soybean WYN341GmC.【Method】Using the event-specific junction sequence of genetically modified soybean WYN341GmC as the target, specific primers and probes were designed, and the reaction system parameters, including primer and probe concentrations, were optimized. The performance of the method was evaluated in terms of specificity, sensitivity, linear range, trueness, and precision. During method screening, five criteria were primarily considered: stable dual-channel droplet signals in the two-dimensional amplitude plots, clear separation between positive and negative droplets, minimal rain, a sufficient number of valid droplets, and an observed copy number ratio of WYN341GmC to Lectin close to the theoretical value. During method validation, droplet digital PCR, combined with serial dilution experiments using reference materials, was mainly employed to assess the limit of quantification (LOQ), limit of detection (LOD), trueness, and precision of the method.【Result】A duplex ddPCR method for precise quantification was established using the event-specific sequence of WYN341GmC and the soybean endogenous reference gene Lectin as detection targets. Based on the comprehensive evaluation of the above screening criteria, the event-specific primer/probe set WYN341LB-QF2/LB-QR1/LB-QP1 and the endogenous reference primer/probe set LectinQF/QR/QP were ultimately selected for subsequent experiments. For the reaction system, the two primer/probe sets were used at the same concentration levels, with a primer concentration of 0.8 μmol·L^-1and a probe concentration of 0.4 μmol·L^-1, and the annealing/extension temperature was optimized to 58 ℃. Under these ddPCR conditions, the transgene and endogenous reference droplets exhibited clear clustering and stable partitioning, and the method showed good precision. Performance validation demonstrated that the LOD of the method was 8 copies and the LOQ was 17 copies, indicating that the method was suitable for reliable quantification. Within the dynamic range of 17 to 2.08×10⁴ copies, the coefficients of determination (R²) of the regression curves were 1.000 for WYN341GmC and 0.999 for Lectin, demonstrating a strong linear relationship between the theoretical and measured template copy numbers and confirming the suitability of the method for quantitative detection within this range. When applied to blind soybean samples, the method enabled accurate quantification of WYN341GmC content, with the bias in value assignment controlled within 25%.【Conclusion】A duplex ddPCR method for the quantification of genetically modified soybean WYN341GmC was established. The detection targets, reaction system, and assay conditions were clearly defined, and the relevant methodological validation was completed. This method can be used on the ddPCR platform for the precise quantification of WYN341GmC event content.

Key words: duplex droplet digital PCR, genetically modified soybean WYN341GmC, accurate quantification, event specificity, method validation

宋可心, 张茜南, 章金益, 孔影, 刘建, 兰青阔, 王永, 吴疆, 赵新. 转基因大豆WYN341GmC数字PCR精准定量检测方法的建立[J]. 中国农业科学, 2026, 59(14): 3022-3037.

SONG KeXin, ZHANG QianNan, ZHANG JinYi, KONG Ying, LIU Jian, LAN QingKuo, WANG Yong, WU Jiang, ZHAO Xin. Establishment of Digital PCR Method for Quantitative Detection of Genetically Modified Soybean WYN341GmC[J]. Scientia Agricultura Sinica, 2026, 59(14): 3022-3037.

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靶标 Target	引物/探针 Primer/probe	序列 Sequence (5′-3′)	片段长度 Fragment size (bp)	参考文献 Reference
WYN341GmC转化体 WYN341GmC event	WYN341LB-QF2	AGTAGGAGGCGATACTTGA	109	待发布国家标准* Forthcoming national standard
	WYN341LB-QR1	AAACGTCCGCAATGTGTTATT
	WYN341LB-QP1	FAM-TCATCCCGTAAACAAATTGACGCTTAGACA-BHQ1
	WYN341LB-QF2	AGTAGGAGGCGATACTTGA	130	自行设计 Self-developed
	WYN341_5BD_QR1	GGCGTTAATTCAGTACATTA
	WYN341LB-QP1	FAM-TCATCCCGTAAACAAATTGACGCTTAGACA-BHQ1
	WYN341LB-QF2	AGTAGGAGGCGATACTTGA	161	自行设计 Self-developed
	GM341_5PE_QR1	ACTCCATGCAAGCTTTCTCACTAA
	WYN341LB-QP1	FAM-TCATCCCGTAAACAAATTGACGCTTAGACA-BHQ1
	GM341LB-F5	TTCTTTGATTAGTAGGAGGCG	119	自行设计 Self-developed
	WYN341LB-QR1	AAACGTCCGCAATGTGTTATT
	WYN341LB-QP1	FAM-TCATCCCGTAAACAAATTGACGCTTAGACA-BHQ1
	GM341LB-F5	TTCTTTGATTAGTAGGAGGCG	140	自行设计 Self-developed
	WYN341_5BD_QR1	GGCGTTAATTCAGTACATTA
	WYN341LB-QP1	FAM-TCATCCCGTAAACAAATTGACGCTTAGACA-BHQ1
	GM341LB-F5	TTCTTTGATTAGTAGGAGGCG	171	自行设计 Self-developed
	GM341_5PE_QR1	ACTCCATGCAAGCTTTCTCACTAA
	WYN341LB-QP1	FAM-TCATCCCGTAAACAAATTGACGCTTAGACA-BHQ1
	WYN341_3BD_QF1	CGCAAACTAGGATAAATTATC	207	自行设计 Self-developed
	WYN341-RB-R2	TGCGGCATGCATCTAAGCAA
	WYN341_3BD_P1	FAM-CGCGGTGTCATCTATGTTACT-BHQ1
Lectin	LectinQF	GCCCTCTACTCCACCCCCA	118	[24]
	LectinQR	GCCCATCTGCAAGCCTTTTT
	LectinQP	FAM/HEX-AGCTTCGCCGCTTCCTTCAACTTCAC-BHQ1
	Lectin1QF	CCAGCTTCGCCGCTTCCTTC	74	[25]
	Lectin1QR	GAAGGCAAGCCCATCTGCAAGCC
	Lectin1QP	FAM/HEX-CTTCACCTTCTATGCCCCTGACAC-TAMRA
	Lectin2QF	GCAAATCTCTGGCCTTT	81	[26]
	Lectin2QR	CTTGCCCGTATTGATGACGTC
	Lectin2QP	HEX-TTCATGTTCGGCGGTCTCGCG-TAMRA
	Lectin3QF	GGACAAAGAAACCGGTAGCGT	89	[27]
	Lectin3QR	GCCCATCTGCAAGCCTTTTT
	Lectin3QP	HEX-AGCTTCGCCGCTTCCTTCAACTTCAC-TAMRA

样品 Sample	成分 Composition
阴性对照（NTC） No-template control	非转基因大豆WYN341GmC Non-GMO soybean WYN341GmC
转化体材料 Transformer material	转基因大豆WYN341GmC Transgenic soybean WYN341GmC
转基因大豆混合样品 GM soybean mix	A2704-12, A5547-127, CV127, DAS68416-4, MON88302, MON87705, MON87769, MON89788, GTS40-3-2, SHZD32-1, FG72, 305423, 356043, 73496
转基因玉米混合样品 GM corn mix	Bt11、GA21、NK603、T25、MIR604、MIR162、双抗12-5、C0030.3.5、C0010.3.7、MON87427、MON89034、MON88017、59122、4114、3272 Bt11, GA21, NK603, T25, MIR604, MIR162, Shuangkang 12-5, C0030.3.5, C0010.3.7, MON87427, MON89034, MON88017, 59122, 4114, 3272
转基因棉花混合样品 GM cotton mix	LLCOTTON25, GHB614, COT102, MON88913, MON531, MON1445, MON15985
转基因油菜混合样品 GM rape mix	Ms1, Ms8, MON88302, oxy235, RF1, RF2, RF3, Topas19/2, T45, 73496
转基因水稻混合样品 GM rice mix	TT51-1、T1C-19、克螟稻、科丰2号、科丰6号、科丰8号、M12、G6H1 TT51-1, T1C-19, Kemingdao, Kefeng 2, Kefeng 6, Kefeng 8, M12, G6H1
非转基因大豆混合样品 Non-GMO soybean mix	中黄13、中黄30、黑农84、合丰55、Williams 82、华春6号、东农50、冀豆17 Zhonghuang 13, Zhonghuang 30, Heinong 84, Hefeng 55, Willians 82, Huachun 6, Dongnong 50, Jidou 17

转化体 Transformer	理论值 Theoretical value (copies/μL)	测量值Measured value (copies/μL)					标准偏差 SD	相对标准偏差 RSD (%)	偏差 Bias (%)
转化体 Transformer	理论值 Theoretical value (copies/μL)	重复1 Replicate 1	重复2 Replicate 2	重复3 Replicate 3	重复4 Replicate 4	平均值 Average	标准偏差 SD	相对标准偏差 RSD (%)	偏差 Bias (%)
WYN341GmC	20800	19983.00	20179.00	20431.00	21382.00	20493.75	619.91	3.02	-1.47
	4160	4010.00	4072.00	4106.00	4095.00	4070.75	42.91	1.05	-2.15
	832	759.00	774.00	801.00	826.00	790.00	29.63	3.75	-5.05
	166	179.00	171.00	168.00	170.00	172.00	4.83	2.81	3.61
	33	31.00	34.00	39.00	30.00	33.50	4.04	12.06	1.52
	17	17.00	16.00	20.00	19.00	18.00	1.83	10.14	5.88
	8	8.20	6.60	12.00	10.00	9.20	2.33	25.29	15.00
	4	3.60	4.50	7.10	6.30	5.38	1.61	29.90	34.50
Lectin	20800	19380.00	19881.00	19933.00	20701.00	19973.75	545.19	2.73	-3.97
	4160	3932.00	4155.00	4197.00	4221.00	4126.25	132.34	3.21	-0.81
	832	764.00	771.00	820.00	863.00	804.50	46.28	5.75	-3.31
	166	180.00	175.00	173.00	177.00	176.25	2.99	1.69	5.92
	33	32.00	33.00	41.00	32.00	34.50	4.36	12.63	3.67
	17	18.00	17.00	19.00	21.00	18.75	1.71	9.11	12.68
	8	8.00	7.80	8.90	13.00	9.43	2.43	25.79	13.34
	4	4.10	6.00	6.60	4.00	5.18	1.32	25.56	24.52
比值 Ratio	20800	1.03	1.01	1.02	1.03	1.02	0.01	0.94	-0.02
	4160	1.02	0.98	0.98	0.97	0.99	0.02	2.25	-0.01
	832	0.99	1.00	0.98	0.96	0.98	0.02	1.74	-0.02
	166	0.99	0.98	0.97	0.96	0.98	0.01	1.32	-0.02
	33	0.97	1.03	0.95	0.94	0.97	0.04	4.15	-0.03
	17	0.94	0.94	1.05	0.90	0.96	0.06	6.73	-0.04
	8	1.03	0.85	1.35	0.77	1.00	0.26	25.74	0.00
	4	0.88	0.75	1.08	1.58	1.07	0.36	33.99	0.07

质量分数 Mass fraction		重复1 Replicate 1	重复2 Replicate 2	重复3 Replicate 3	重复4 Replicate 4	平均值 Average	标准偏差 SD	相对标准偏差 RSD (%)	偏差 Bias (%)
5% (2080 copies/μL)	WYN341GmC	2022	2117	2100	2033	2068	47.49	2.30	-0.58
	Lectin	41040	41428	41739	40198	41101	666.61	1.62	-1.20
	转基因DNA含量 Transgenic DNA content	4.93	5.11	5.03	5.06	5.03	0.08	1.51	0.60
1% (416 copies/μL)	WYN341GmC	463	442	421	437	441	17.33	3.93	6.01
	Lectin	42589	41932	40584	41978	41771	845.94	2.03	0.41
	转基因DNA含量 Transgenic DNA content	1.09	1.05	1.04	1.04	1.06	0.02	2.26	6.00
0.1% (41.6 copies/μL)	WYN341GmC	39	44	40	37	40	2.94	7.36	-3.85
	Lectin	40595	39085	40682	39340	39926	830.62	2.08	-4.02
	转基因DNA含量 Transgenic DNA content	0.10	0.08	0.10	0.09	0.09	0.01	10.35	-10.00