Scientia Agricultura Sinica ›› 2026, Vol. 59 ›› Issue (1): 29-40.doi: 10.3864/j.issn.0578-1752.2026.01.003

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Development and Validation of Event-Specific PCR Method for the Quantification of Genetically Modified Soybean DBN8205

WU Qiong1(), XIE XiangTing2, WANG Lei2, MOU Yong2, LI JinWei1,*()   

  1. 1 Beijing Chuangzhong Technology Co., Ltd., Beijing 100194
    2 Beijing Dabeinong Biotechnology Co., Ltd., Beijing 100194
  • Received:2025-07-23 Accepted:2025-10-02 Online:2026-01-01 Published:2026-01-07
  • Contact: LI JinWei

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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

Table 1

Primers and probes for DBN8205 event and Lectin reference gene"

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

Fig. 1

Screening and specificity testing of event- specific primer/probe combinations a: Selection of the optimal primer/probe combination; b: Positions of primers and probes on the junction between T-DNA and flanking genomic DNA, where 1-32 bp represents the flanking genomic sequence in uppercase letters, and 33-120 bp represents the inserted T-DNA sequence in lowercase letters, primer sequences are highlighted by underlined letters, and the probe sequence is indicated by blue letters with a wavy underline; c: Specificity testing of the primer/probe combination DBN8205-QF/QR/QP"

Table 2

Standard curves of DBN8205 event and Lectin reference gene"

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
lectin -3.302 100.80 0.997
Rep2 DBN8205 -3.341 99.20 0.998
lectin -3.277 101.90 0.998
Rep3 DBN8205 -3.394 97.10 0.999
lectin -3.445 95.10 0.999

Table 3

LOQ testing of DBN8205 event-specific qPCR method"

重复
Replicate		 S5（100 copies, 0.25%） S6（40 copies, 0.10%） S7（20 copies, 0.05%） S8 (10 copies, 0.025%)
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
Biasr (%)		 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

Table 4

Accuracy analysis of DBN8205 event-specific quantitative PCR method"

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

Fig. 2

Optimization of primer/probe concentration and annealing temperature for DBN8205/Lectin duplex ddPCR a: One-dimension (1-D) plots of the DBN8205 eventacross an annealing temperature gradient from 58 to 62 ℃; b: Lectin reference gene across an annealing temperature gradient from 58 to 62 ℃; c: The corresponding DBN8205/Lectin copy number ratios measured at each temperature; d: 1-D plots of the DBN8205 event for optimization of primer/probe concentration; e: 1-D plots of the Lectin reference gene for optimization of primer/probe concentration; f: The resulting DBN8205/Lectin copy number ratios under different concentration schemes, where 1-4, identical concentrations for both assays (100/50, 200/100, 400/200, 800/400 nmol·L-1), 5-8, different concentrations for DBN8205 (400/200, 200/100, 400/200, 800/400 nmol·L-1) and Lectin (200/100, 400/200, 800/400, 400/200 nmol·L-1); g: 1-D plots of the DBN8205 event under the final optimized reaction conditions; h: 1-D plots of the Lectin reference gene under the final optimized reaction conditions; i: 2-D plot of DBN8205 event and Lectin reference gene under the final optimized reaction conditions"

Table 5

Measured copy number and ratio of serial diluted DNA solution by the DBN8205/Lectin duplex ddPCR"

靶标
Target		 预期模板量
Expected amount (copy/reaction)		 测量值 Measured value (copy/reaction )
Rep1 Rep2 Rep3 均值
Mean		 标准差
SD		 相对标准差
RSD (%)		 相对偏倚
Biasr (%)
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

Fig. 3

Dynamic range of DBN8205/Lectin duplex ddPCR a: Linearity of the duplex ddPCR assay for DBN8205 with a dynamic range of 40 to 8.0×10⁴ copies/reaction; b: Lectin with a dynamic range of 40 to 8.0×10⁴ copies/reaction; c: The measured DBN8205/Lectin copy number ratios across different template quantities"

Table 6

The statistical analysis of collaborative validation results of eight laboratories"

样品
Samples		 预期值 Expected value (%)
5.0 3.0 1.0 0.5 0.1
返回数据实验室数Labs reporting results 8 8 8 8 8
每个实验室平行样品个数Replicate 3 3 3 3 3
离群值数Replicate 0 1 0 0 0
平均值Average (%) 5.583 3.292 1.102 0.528 0.107
重复性标准差sr (%) 0.201 0.117 0.109 0.036 0.009
重复性相对标准差RSDr (%) 3.599 3.547 9.852 6.751 8.241
再现性标准差sR (%) 0.337 0.177 0.124 0.046 0.008
再现性相对标准差RSDR (%) 6.028 5.391 11.205 8.741 7.920
偏倚Bias (%) 0.583 0.292 0.102 0.028 0.007
相对偏倚Biasr (%) 11.660 9.742 10.224 5.695 7.297
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