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

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

• 技术与方法 • 上一篇

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

闫家通¹^,²(), 陈冠玮¹^,², 缪青梅², 彭城², 杨蕾², 陈笑芸², 徐晓丽², 魏巍², 徐俊锋²(), 汪小福²()

1.浙江师范大学生命科学学院，金华 321004
2.浙江省农业科学院，农产品质量安全全国重点实验室，农业农村部农业转基因生物溯源重点实验室，全省作物种质创新与利用重点实验室，杭州310021

收稿日期:2024-11-07 修回日期:2024-12-16 出版日期:2025-06-20 发布日期:2025-02-24
通讯作者: 徐俊锋，博士，研究员，研究方向：转基因安全评价。E-mail: njjfxu@163.com;
汪小福，博士，研究员，研究方向：转基因安全评价。E-mail: yywxf1981@163.com
作者简介:闫家通，硕士研究生，专业方向：生物化学与分子生物学。E-mail: yjt15150101315@163.com
基金资助:
浙江省自然科学基金重点项目(LZ23D030001);农业生物育种重大项目(2022ZD0401908);浙江省科技创新领军人才项目(2021R52046)

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 Published:2025-06-20 Online:2025-02-24
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)

摘要/Abstract

摘要：

随着我国转基因玉米和转基因大豆的商业化种植，为了保证我国生物育种产业的健康发展，迫切需要开发转基因玉米和大豆的现场快速检测技术。本研究针对转基因玉米和大豆现场快速检测技术的不足，提出了一种基于重组酶聚合酶扩增技术(recombinase polymerase amplification，RPA)的快速检测方法。选择具有代表性的转基因玉米(双抗12-5、DBN9936、MON810)和大豆(DBN9004、GTS40-3-2、SHZD32-1)为检测对象，根据其侧翼序列，筛选特异性引物和探针，建立了高特异性、灵敏度达20拷贝的RPA扩增体系。为了快速获取DNA，种子样品利用核酸释放剂获得可用于RPA扩增的模板，为了消除叶片中叶绿素并快速获取DNA，叶片样品利用本实验室研发的核酸快速一体化提取装置快速获取DNA，以消除叶绿素干扰。结合便携式金属浴启动RPA扩增，蓝光切胶仪用于可视化检测，构建了转基因玉米和大豆的现场快速检测方法。该方法检测时间约为20 min，与常规PCR检测结果一致，适用于现场快速检测，无需大型设备，具备较强的实用性和适应性。该方法为转基因作物现场检测提供了技术支持，并为其他核酸快速检测技术的应用提供参考。

关键词: 转基因作物, RPA反应, 荧光可视化, 现场检测, 快速提取

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

闫家通, 陈冠玮, 缪青梅, 彭城, 杨蕾, 陈笑芸, 徐晓丽, 魏巍, 徐俊锋, 汪小福. 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[J]. 遗传, 2025, 47(6): 694-707.

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.

图/表 7

表1

本文所用引物与探针序列"

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]

表1

图1

图2

图3

图4

图5

图6

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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

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