基于重组酶聚合酶扩增的转基因玉米和大豆现场快速检测方法

doi:10.16288/j.yczz.24-322

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Hereditas(Beijing) ›› 2025, Vol. 47 ›› Issue (6): 694-707.doi: 10.16288/j.yczz.24-322

• Technique and Method • Previous Articles

Onsite rapid detection method for genetically modified maize and soybean based on recombinase polymerase amplification

Jiatong Yan¹^,²(), Guanwei Chen¹^,², Qingmei Miao², Cheng Peng², Lei Yang², Xiaoyun Chen², Xiaoli Xu², Wei Wei², Junfeng Xu²(), Xiaofu Wang²()

1. College of Life Science, Zhejiang Normal University, Jinhua 321004, China
2. State Key Laboratory for Quality and Safety of Agro-products, Key Laboratory of Traceability for Agricultural Genetically Modified Organisms, Ministry of Agriculture and Rural Affairs of China, Zhejiang Key Laboratory of Crop Germplasm Innovation and Utilization, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, China

Received:2024-11-07 Revised:2024-12-16 Online:2025-06-20 Published:2025-02-24
Contact: Junfeng Xu, Xiaofu Wang E-mail:yjt15150101315@163.com;njjfxu@163.com;yywxf1981@163.com
Supported by:
Zhejiang Province Natural Science Foundation Key Project(LZ23D030001);Major project of Agricultural Biological Breeding(2022ZD0401908);Zhejiang Province Science and Technology Innovation Leading Talent Project(2021R52046)

Abstract

Abstract:

With the commercial planting of transgenic maize and transgenic soybeans in China, in order to ensure the healthy development of China’s biological breeding industry, it is urgent to develop on-site rapid detection techniques for transgenic maize and soybeans. In this study, a rapid detection method based on recombinase polymerase amplification (RPA) was proposed to overcome the shortcomings of on-site rapid detection of transgenic maize and soybean. Representative transgenic maize (Shuang Kang 12-5, DBN9936, MON810) and soybean (DBN9004, GTS40-3-2, SHZD32-1) were selected as test objects. According to their flanking sequence, specific primers and probes were designed and screened, and a RPA amplification system with high specificity and sensitivity of 20 copies for each transgenic event was established. In order to quickly obtain DNA, seed samples use nucleic acid release agents to obtain templates that can be used for RPA amplification. In order to eliminate the interference of chlorophyll in leaves on RPA fluorescence signal and quickly obtain DNA, leaf samples use the nucleic acid rapid integrated extraction device developed by our laboratory to quickly obtain DNA and eliminate the chlorophyll interference. Combined with a portable metal bath for initiating RPA amplification, a blue light gel cutter for visual detection, and a rapid on-site detection method for transgenic maize and soybean was constructed. The entire detection process required approximately 20 minutes, and the detection result was consistent with the result of conventional PCR. It is suitable for on-site rapid detection without large equipment, and has strong practicability and adaptability. The proposed approach provides technical support for on-site detection of transgenic crops and offers a reference for the application of other rapid nucleic acid detection technologies.

Key words: genetically modified crops, RPA reaction, fluorescence visualization, field detection, rapid extraction

Jiatong Yan, Guanwei Chen, Qingmei Miao, Cheng Peng, Lei Yang, Xiaoyun Chen, Xiaoli Xu, Wei Wei, Junfeng Xu, Xiaofu Wang. Onsite rapid detection method for genetically modified maize and soybean based on recombinase polymerase amplification[J]. Hereditas(Beijing), 2025, 47(6): 694-707.

Figures/Tables 7

Table 1

Primer sequences and probe sequences used in this study"

目的	引物名称	引物序列(5ʹ→3ʹ)	参考文献
荧光RPA	12-5-F1	TCCGACGCCTCGACGCTCGTGGTGACTCGC	本研究
	12-5-F2	CGACCAAGTCATGAAGAACTCCCACTGCCG
	12-5-F3	CGACCCGAAGATGGAGGCCTACTGCGATGA
	12-5-F4	GACGTTCGGTGCCTGGAAGACAAGTTCTAC
	12-5-F5	CGTCATCGACCAAGTCATGAAGAACTCCCA
	12-5-F6	CAGCTCGTCATCGACCAAGTCATGA
	12-5-F7	CCGACGCCTCGACGCTCGTGGTGACTCGCAGC
	12-5-F8	CATCGACCAAGTCATGAAGAACTCCCACTGCCG
	12-5-R1	ATGCTAGAGCAGCTTGAGCTTGGATCAGAT
	12-5-R2	CCGATCGCCCTTCCCAACAGTTGCGCAGCC
	12-5-R3	CCAGCTGGCGTAATAGCGAAGAGGCCCGCA
	12-5-R4	TAATAGCGAAGAGGCCCGCACCGATCGCCC
	12-5-R5	TGAATGGCGAATGCTAGAGCAGCTTGAGCT
	12-5-R6	CTGAATGGCGAATGCTAGAGCAGCT
	12-5-R7	CTTGAGCTTGGATCAGATTGTCGT TTCCCGCC
	12-5-R8	CCCAACAGTTGCGCAGCCTGAATGGCGAATGC
	12-5-P	AACCACATCGCCCGACGCTACAACGAGAC[FAM-dT][THF]A [BHQ1-dT]AGTTTAAACTGAA[3′-block]
	DBN9936-F1	TTAAGTTGTCTAAGCGTCAATTGGATCCCG
	DBN9936-F2	AAACCTAATCAGTCAGTGCCGGTGAGAGCG
	DBN9936-F3	AAGCGGCCTCGTCAGCCAACACCGTGAGGC
	DBN9936-F4	AGTCAGTGCCGGTGAGAGCGTAGCTGCCTG
	DBN9936-F5	TATTAAGTTGTCTAAGCGTCAATTGGATC
	DBN9936-F6	GGTCCCGTTTAAACCTAATCAGTCAGTGCC
	DBN9936-F7	GGTGAGAGCGTAGCTGCCTGGAAGCGGCCT
	DBN9936-F8	GAAGCGGCCTCGTCAGCCAACACCGTGAGG
	DBN9936-R1	ATATTTATGGGCCATAAACAAGGACCGGCG
	DBN9936-R2	AATAATCGTCTTGTACTACTGTGCTGAGGG
	DBN9936-R3	AATAGGAAACAGGAAAGGAGACCCGCGCGC
	DBN9936-R4	CGTCTTGTACTACTGTGCTGAGGGGCGTGC
	DBN9936-R5	GCAAATAGGAAACAGGAAAGGAGACCCGCG
	DBN9936-R6	CAATTGTTTAAAAATAATAATCGTCTTGTA
	DBN9936-R7	ATAAACAAGGACCGGCGGTCAAGTGAGTGC
	DBN9936-R8	CATGTCATCCGGAAGCAGGCCGATATTTAT
	DBN9936-P	TATCAGTCAAACGGTCCCGTTTAAACTA[FAM-dT]C[THF]G [BHQ1-dT]GTTTGAAAGCGAG[3′-block]
	MON810-F1	TCAACGTGCCCGGTACTGGTTCCCTCTGGC
	MON810-F2	CGCTGAGCGCCCCCAGCCCGATCGGCAAGT
	MON810-F3	GTGCCCACCACAGCCACCACTTCTCCTTGG
	MON810-F4	CAGCCACCACTTCTCCTTGGACATCGATGT
	MON810-R1	GCTGCAGGTGGTCTTACATCTAAGAAGGAT
	MON810-R2	TTCTCATGATGAAAGCACTAGTACTGCCAA
	MON810-R3	CAAAAGGACCTGACTGCTCGCAAGCAAATT
	MON810-R4	GTCTTACATCTAAGAAGGATTCTCATGATG
	MON810-P	CTGCACCGACCTGAACGAGGACTTTCGG[FAM-dT]A[THF]CC [BHQ1-dT]TCTTTCATTTCC[3′-block]
	DBN9004-F1	TCATGTGTTGAGCATATAAGAAACCCTTAG
	DBN9004-F2	TTCTAATTCCTAAAACCAAAATCCAGTGGC
	DBN9004-F3	AACCAAAATCCAGTGGCGCGCCCATGATGG
	DBN9004-F4	ATGGCCGTATCCGCAATGTGTTATTAAGTT
	DBN9004-R1	ACTTGATGAAAGAAGAGCGTTGCATTTACG
	DBN9004-R2	AGAAGAGCGTTGCATTTACGCTCCATAAAC
	DBN9004-R3	AGAGCGTTGCATTTACGCTCCATAAACGTG
	DBN9004-R4	TTGATGAAAGAAGAGCGTTGCATTTACGCTCC
	DBN9004-P	GTTGTCTAAGCGTCAATTTGTTTACAAC[FAM-dT]C[THF]G [BHQ1-dT]GACCCCGTAATGA[3′-block]
	GTS40-3-2-F1	AACCGATGCTAATGAGTTATTTTTGCATGC
	GTS40-3-2-F2	TCATACATACAGGTTAAAATAAACATAGGG
	GTS40-3-2-F3	TATTTTTGCATGCTTTAATTTGTTTCTATC
	GTS40-3-2-R1	ATAGTGGGATTGTGCGTCATCCCTTACGTC
	GTS40-3-2-R2	TCAGTGGAGATATCACATCAATCCACTTGC
	GTS40-3-2-R3	TTGCTTTGAAGACGTGGTTGGAACGTCTTC
	GTS40-3-2-R4	TTCCACGATGCTCCTCGTGGGTGGGGGTCC
	GTS40-3-2-R5	ACTGTCGGCAGAGGCATCTTGAACGATAGC
	GTS40-3-2-P	CATAGGGAACCCAAATGGAAAAGGAAGG[6FAM-dT]G[THF]C [BHQ1-dT]CCTACAAATGCCA[3′-block]
	SHZD32-1-F1	CTACACATCCCCTTCCCCCTTCGCTGTTGC
	SHZD32-1-F2	CCTTCGCTGTTGCAACTCATTGCACAAAGACC
	SHZD32-1-F3	CAAAGTCACTCATTATTGGAACCCTACACATC
SHZD32-1-F4	CGTCTATTTCATTAACTTTAGGATGTTGCTAA
SHZD32-1-R1	CTTAGTATGTATTTGTATTTGTAAAATACTTC
SHZD32-1-R2	CTTCTATCAATAAAATTTCTAATTCCTAAAAC
SHZD32-1-R3	TAAAATTTCTAATTCCTAAAACCAAAATCCAG
SHZD32-1-R4	AGGGTTCCTATAGGGTTTCGCTCATGTGTTGA
SHZD32-1-P	TGTTGCTAAGCACATGCATTTTAACGAA[FAM-dT]T[THF]AT [BHQ1-dT]CGGGGGATCTGG[3′-block]
普通PCR	12-5-F	CAACGTCGTGACTGGGAAAA	[20]
	12-5-R	TGGAAGACAAGTTCTACGGGCT	[20]
	DBN9936-F	CTCGTCAGCCAACACCGTGA	[21]
	DBN9936-R	CCCCGCCTCATCTTGATCCA	[21]
	MON810-F	ACCTCGAGATTTACCTGATCCG	[22]
	MON810-R	TGCTGCAGGTGGTCTTACATC	[22]
	DBN9004-F	CGGGACTTGATGAAAGAAGAG	待颁布
	DBN9004-R	GGTTTCGCTCATGTGTTGAG	待颁布
	GTS40-3-2-F	TTCAAACCCTTCAATTTAACCGAT	[23]
	GTS40-3-2-R	AAGGATAGTGGGATTGTGCGTC	[23]
	SHZD32-1-F	GAGCAGCTTGAGCTTGGA	[24]
	SHZD32-1-R	CGAATTTCACCAAAACACTAA	[24]

Table 1

Fig. 1

Fig. 2

Fig. 3

Fig. 4

Fig. 5

Fig. 6

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Onsite rapid detection method for genetically modified maize and soybean based on recombinase polymerase amplification

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[1]	Yu Chen, Xiaoyun Chen, Cheng Peng, Junfeng Xu, Jie Shen, Yueying Li, Xiaofu Wang. Establishment of a field visual detection method for genetically modified maize ‘Shuangkang’12-5 by fluorescence RPA [J]. Hereditas(Beijing), 2021, 43(8): 802-812.
[2]	. Unintended effects assessment of genetically modified crops using omics techniques [J]. HEREDITAS, 2013, 35(12): 1360-1367.