Scientia Agricultura Sinica ›› 2024, Vol. 57 ›› Issue (23): 4698-4711.doi: 10.3864/j.issn.0578-1752.2024.23.010

• PLANT PROTECTION • Previous Articles     Next Articles

Accurate and Rapid Identification of Event Purity for Transgenic Soybean Seeds Based on Duplex Real-Time Fluorescence PCR Method

LI YunJing1(), REN XueZhen2, XIAO Fang1, JIN Fang2, GAO HongFei1, JING Qi2, WU YuHua1, SUN Quan2, LI Jun1, WANG Pei2, ZHAI ShanShan1, JIN ShiQiao2(), WU Gang1()   

  1. 1 Oil Crops Research Institute of Chinese Academy of Agricultural Sciences/Key Laboratory of Biology and Genetic Improvement of Oil Crops, Ministry of Agriculture and Rural Affairs/Inspection and Testing Center (Wuhan) for Plant Ecological Environment Safety, Ministry of Agriculture and Rural Affairs/Key Laboratory of Agricultural Genetically Modified Organisms Traceability, Ministry of Agriculture and Rural Affairs, Wuhan 430062
    2 National Agro-Tech Extension and Service Center, Beijing 100125
  • Received:2024-07-03 Accepted:2024-09-16 Online:2024-12-01 Published:2024-12-07

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Abstract:

【Objective】 Production and application safety certificates have been successively granted to herbicide-tolerant soybean, including SHZD3201, DBN9004, and ZH10-6. The objective of this study is to establish duplex real-time fluorescence PCR detection methods for these three events, which are applied to the accurate and rapid identification of the purity of soybean transgenic event seeds, and to provide technical support for the quality and safety supervision of soybean transgenic varieties seeds.【Method】 Three event-specific real-time PCR methods for herbicide-tolerant soybean: SHZD3201, DBN9004, ZH10-6, and four soybean-specific real-time PCR methods targeting the Lectin were collected. Through the comparison of ΔCt values, the most effective soybean reference gene detection approach for duplex real-time fluorescence PCR was chosen. The study further adjusted the concentration of primers and probes for both events and Lectin, optimizing the reaction system of duplex real-time fluorescence PCR. The duplex real-time fluorescence PCR methods underwent specificity testing using diverse types of samples. The limit of detection (LOD) was assessed using range of gradient samples containing 2 000, 200, 20, 10, 5, and 1 copies. Using one-step extraction solution for rapid grinding of individual seeds, the diluted crude extract was directly used for duplex real-time fluorescence PCR amplification, thereby conducting event purity testing of individual seeds for herbicide-tolerant soybeans SHZD3201, DBN9004, and ZH10-6.【Result】 Following the determination of the minimum ΔCt value through testing and calculation, the optimal soybean reference gene for detecting Lectin was selected in duplex real-time fluorescence PCR of SHZD3201, DBN9004, and ZH10-6 events. After optimization, the SHZD3201/Lectin duplex real-time fluorescence PCR reaction system exhibited optimal performance with the primer/probe concentration of 0.3 μmol·L-1/0.15 μmol·L-1 for SHZD3201 event and the primer/probe concentration of 0.3 μmol·L-1/0.15 μmol·L-1 for Lectin. In the DBN9004/Lectin duplex real-time fluorescence PCR reaction system, the primer/probe concentrations for both the DBN9004 event and Lectin were identical, setting at 0.4 μmol·L-1 for the prime and 0.2 μmol·L-1 for the probe. In the duplex real-time fluorescence PCR reaction system of ZH10-6/Lectin, the primer/probe concentration of ZH10-6 event was 0.4 μmol·L-1/0.2 μmol·L-1, the primer/probe concentration for the Lectin was 0.5 μmol·L-1/0.25 μmol·L-1. The three methods had good specificity with the LOD of 10 copies each. Using 100 simulated single-seed samples, after rapid grinding and a 5-fold dilution of the template, the direct duplex real-time fluorescence PCR was used for rapid amplification, and the event purity of transgenic seeds of SHZD3201, DBN9004, and ZH10-6 was successfully detected.【Conclusion】 This study successfully established three duplex real-time fluorescence PCR methods for herbicide-tolerant soybean SHZD3201, DBN9004, and ZH10-6. By combining a rapid DNA extraction method using one-step extraction solution, the research achieved precise and rapid detection of event purity in transgenic soybean seeds.

Key words: herbicide-tolerant soybean, seed, duplex real-time fluorescence PCR, event purity

Table 1

Materials information"

样品Sample 组分Component
转基因大豆Transgenic soybean GTS40-3-2, MON89788, A5547-127, A2704-12, 356043, 305423, CV127, MON87701, MON87708, MON87769, MON87705, FG72, DAS81419-2
转基因玉米Transgenic maize Bt11, Bt176, MON810, MON863, GA21, NK603, T25, TC1507, MON89034, MON88017, 59122, MIR604, 3272, MON87460, DAS40278-9, 4114, MON87427, 5307
转基因水稻Transgenic rice TT51-1, KF-6, KMD-1, M12, KF-8, KF-2, G6H1, T1C-19
转基因棉花Transgenic cotton MON1445, MON531, MON15985, LLCOTTON25, MON88913, GHB614, COT102
转基因油菜Transgenic rapeseed MS1, MS8, RF1, RF2, RF3, T45, Oxy-235, Topas19/2, MON88302, 73496

Table 2

Information of primers and probes"

名称
Name		 引物/探针名称
Primer/probe name		 序列
Sequence (5′-3′)		 扩增片段
Amplified fragment (bp)		 来源
Source
SHZD3201 SHZD32-1QF TCGTTTCCCGCCATAAGG 120 [27]
SHZD32-1QR CATCAACCAAGAGCAACAGCAT
SHZD32-1QP FAM-TCCGACCACCACGAGACCGTAGTACA-BHQ1
DBN9004 DBN9004-QF AACGCGGCCGCTCTAG 165 [28]
DBN9004-QR ATTATGTGGAAAGAAATGACCGAAA
DBN9004-QP FAM-CGCCGGGTCCCGTTTAAACTATCAGT-BHQ1
中黄6106
ZH10-6		 ZH6106-QF TCTAAGCGTCAATTTGTTTACATCA 96 研发者
Developer
ZH6106-QR AAAGGTGTGAAATGTGAACGC
ZH6106-QP FAM-TTACCTAAAACATCTCAGCACATCGC-BHQ1
Lectin1 lecF CCAGCTTCGCCGCTTCCTTC 74 [35]
lecR GAAGGCAAGCCCATCTGCAAGCC
lecP HEX-CTTCACCTTCTATGCCCCTGACAC-BHQ1
Lectin2 LecF1 TCCACCCCCATCCACATTT 81 [36]
LecR1 GGCATAGAAGGTGAAGTTGAAGGA
LecP1 HEX-AACCGGTAGCGTTGCCAGCTTCG-BHQ1
Lectin3 LecF2 GCCCTCTACTCCACCCCCA 118 [37]
LecR2 GCCCATCTGCAAGCCTTTTT
LecP2 HEX-AGCTTCGCCGCTTCCTTCAACTTCAC-BHQ1
Lectin4 LecF3 TGGTCGCGCCCTCTACTC 70 [38]
LecR3 GGCGAAGCTGGCAACG
LecP3 HEX-CTACCGGTTTCTTTGTCCCAAATGTGGAT-BHQ1

Table 3

Combinations of primer/probe in a duplex real-time fluorescence PCR reaction system"

编号
No.		 转化体
Event		 Lectin
1 0.3 μmol·L-1/0.15 μmol·L-1 0.3 μmol·L-1/0.15 μmol·L-1
2 0.3 μmol·L-1/0.15 μmol·L-1 0.4 μmol·L-1/0.2 μmol·L-1
3 0.3 μmol·L-1/0.15 μmol·L-1 0.5 μmol·L-1/0.25 μmol·L-1
4 0.4 μmol·L-1/0.2 μmol·L-1 0.3 μmol·L-1/0.15 μmol·L-1
5 0.4 μmol·L-1/0.2 μmol·L-1 0.4 μmol·L-1/0.2 μmol·L-1
6 0.4 μmol·L-1/0.2 μmol·L-1 0.5 μmol·L-1/0.25 μmol·L-1
7 0.5 μmol·L-1/0.25 μmol·L-1 0.3 μmol·L-1/0.15 μmol·L-1
8 0.5 μmol·L-1/0.25 μmol·L-1 0.4 μmol·L-1/0.2 μmol·L-1
9 0.5 μmol·L-1/0.25 μmol·L-1 0.5 μmol·L-1/0.25 μmol·L-1

Table 4

Amplification results of events and Lectin in duplex real-time fluorescence PCR"

靶标
Target		 转化体的Ct值Ct value of event Lectin的Ct值Ct value of Lectin ΔCt值
ΔCt value
平行1
Parallel 1		 平行2
Parallel 2		 平行3
Parallel 3		 平均值
Mean		 平行1
Parallel 1		 平行2
Parallel 2		 平行3
Parallel 3		 平均值
Mean
SHZD3201/Lectin1 28.54 28.47 28.49 28.50 29.12 29.09 29.07 29.09 -0.59
SHZD3201/Lectin2 28.48 28.53 28.48 28.50 28.50 28.56 28.40 28.49 0.01
SHZD3201/Lectin3 28.60 28.55 28.52 28.56 29.50 29.49 29.43 29.47 -0.91
SHZD3201/Lectin4 28.75 28.79 28.64 28.73 29.37 29.25 29.34 29.32 -0.59
DBN9004/Lectin1 28.19 28.09 28.19 28.16 29.34 29.11 29.16 29.20 -1.05
DBN9004/Lectin2 28.10 28.06 27.94 28.03 28.37 28.52 28.53 28.47 -0.44
DBN9004/Lectin3 28.08 28.04 28.18 28.10 29.42 29.34 29.48 29.41 -1.31
DBN9004/Lectin4 28.15 28.15 28.18 28.16 29.11 29.09 29.16 29.12 -0.96
中黄6106/Lectin1
ZH10-6/Lectin1		 28.45 28.16 28.13 28.25 29.50 29.26 29.30 29.35 -1.11
中黄6106/Lectin2
ZH10-6/Lectin2		 28.20 28.13 28.08 28.14 28.57 28.39 28.44 28.47 -0.33
中黄6106/Lectin3
ZH10-6/Lectin3		 28.13 28.10 28.13 28.12 29.56 29.48 29.55 29.53 -1.41
中黄6106/Lectin4
ZH10-6/Lectin4		 28.10 27.94 28.18 28.07 29.20 29.10 29.41 29.24 -1.16

Table 5

Optimization of duplex real-time fluorescence PCR reaction system for SHZD3201/Lectin"

编号
No.		 SHZD3201转化体的Ct值Ct value of event SHZD3201 Lectin的Ct值Ct value of Lectin ΔCt值
ΔCt value
平行1
Parallel 1		 平行2
Parallel 2		 平行3
Parallel 3		 平均值
Mean		 平行1
Parallel 1		 平行2
Parallel 2		 平行3
Parallel 3		 平均值
Mean
1 29.19 29.18 29.12 29.16 29.15 29.06 29.07 29.09 0.07
2 29.05 28.89 28.94 28.96 28.56 28.47 28.65 28.56 0.40
3 29.09 29.03 29.02 29.05 28.45 28.50 28.52 28.49 0.56
4 28.81 28.86 28.68 28.78 28.84 28.97 28.85 28.89 -0.10
5 28.83 28.97 28.67 28.82 28.61 28.61 28.60 28.61 0.22
6 29.05 29.04 28.93 29.01 28.60 28.45 28.52 28.52 0.48
7 28.94 28.89 28.81 28.88 29.10 29.05 29.04 29.06 -0.18
8 29.23 29.24 28.98 29.15 29.04 29.01 28.90 28.98 0.17
9 28.73 29.03 28.94 28.90 28.56 28.60 28.61 28.59 0.31

Table 6

Optimization of duplex real-time fluorescence PCR reaction system for DBN9004/Lectin"

编号
No.		 DBN9004转化体的Ct值Ct value of event DBN9004 Lectin的Ct值Ct value of Lectin ΔCt值
ΔCt value
平行1
Parallel 1		 平行2
Parallel 2		 平行3
Parallel 3		 平均值
Mean		 平行1
Parallel 1		 平行2
Parallel 2		 平行3
Parallel 3		 平均值
Mean
1 28.65 28.71 28.63 28.66 28.87 28.99 28.77 28.88 -0.21
2 28.63 28.53 28.50 28.55 28.38 28.31 28.31 28.33 0.22
3 28.55 28.59 28.53 28.56 28.18 28.17 28.09 28.15 0.41
4 28.44 28.36 28.50 28.43 28.44 28.46 28.54 28.48 -0.05
5 28.40 28.39 28.36 28.38 28.26 28.25 28.29 28.27 0.12
6 28.34 28.30 28.45 28.36 28.11 28.07 28.11 28.10 0.27
7 28.55 28.47 28.49 28.50 28.79 28.73 28.68 28.73 -0.23
8 28.79 28.8 28.56 28.72 28.70 28.56 28.45 28.57 0.15
9 28.32 28.53 28.37 28.41 28.10 28.18 28.06 28.11 0.29

Table 7

Optimization of duplex real-time fluorescence PCR reaction system for ZH10-6/Lectin"

编号
No.		 中黄6106转化体的Ct值Ct value of event ZH10-6 Lectin的Ct值Ct value of Lectin ΔCt值
ΔCt value
平行1
Parallel 1		 平行2
Parallel 2		 平行3
Parallel 3		 平均值
Mean		 平行1
Parallel 1		 平行2
Parallel 2		 平行3
Parallel 3		 平均值
Mean
1 28.82 28.77 28.63 28.74 29.31 29.29 29.16 29.25 -0.51
2 28.97 28.79 28.67 28.81 28.82 28.73 28.66 28.74 0.07
3 28.70 28.62 28.50 28.61 28.68 28.41 28.27 28.45 0.15
4 28.59 28.49 28.34 28.47 29.06 29.02 28.78 28.95 -0.48
5 28.59 28.47 28.34 28.47 28.85 28.77 28.65 28.76 -0.29
6 28.51 28.54 28.58 28.54 28.48 28.58 28.57 28.54 0
7 28.52 28.23 28.16 28.30 29.18 29.02 29.02 29.07 -0.77
8 28.41 28.27 28.65 28.44 28.85 28.8 28.95 28.87 -0.42
9 28.84 28.93 29.12 28.96 28.94 28.89 28.91 28.91 0.05

Fig. 1

SHZD3201/Lectin duplex real-time fluorescence PCR specificity test"

Fig. 2

DBN9004/Lectin duplex real-time fluorescence PCR specificity test"

Fig. 3

ZH10-6/Lectin duplex real-time fluorescence PCR specificity test"

Fig. 4

Amplification of event purity detection for SHZD3201 seed"

Fig. 5

Amplification of event purity detection for DBN9004 seed"

Fig. 6

Amplification of event purity detection for ZH10-6 seed"

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