基于DNAzyme信号放大的转基因大豆DBN9004快速检测方法

doi:10.3864/j.issn.0578-1752.2026.02.002

中国农业科学 ›› 2026, Vol. 59 ›› Issue (2): 239-249.doi: 10.3864/j.issn.0578-1752.2026.02.002

• 作物遗传育种·种质资源·分子遗传学 • 上一篇下一篇

基于DNAzyme信号放大的转基因大豆DBN9004快速检测方法

符丽锦¹^,²(), 陈冠玮²^,³(), 肖功²^,³, 汪小福², 彭城², 陈笑芸², 徐俊锋¹^,², 陈子言⁴(), 杨蕾²()

¹ 浙江农林大学现代农学院，杭州 311300
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
³ 浙江师范大学生命科学学院，浙江金华 321004
⁴ 农业农村部科技发展中心，北京 100176

收稿日期:2025-07-24 接受日期:2025-09-22 出版日期:2026-01-16 发布日期:2026-01-22
通信作者:
陈子言，E-mail：moachenzy@163.com。
杨蕾，E-mail：yanglei9203@163.com
联系方式: 符丽锦，E-mail：18389963101@163.com。陈冠玮，E-mail：zkywxs@163.com。符丽锦和陈冠玮为同等贡献作者。
基金资助:
农业生物育种国家科技重大专项(2022ZD0402006); 浙江省自然科学基金(LMS25C200002); 浙江省自然科学基金重点项目(LZ23D030001)

A Rapid Detection Method for Genetically Modified Soybean Dbn9004 Based on Dnazyme Signal Amplification

FU LiJin¹^,²(), CHEN GuanWei²^,³(), XIAO Gong²^,³, WANG XiaoFu², PENG Cheng², CHEN XiaoYun², XU JunFeng¹^,², CHEN ZiYan⁴(), YANG Lei²()

¹ College of Modern Agriculture, Zhejiang A & F University, Hangzhou 311300
² Zhejiang Academy of Agricultural Sciences/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/Zhejiang Key Laboratory of Crop Germplasm Innovation and Utilization, Hangzhou 310021
³ College of Life Science, Zhejiang Normal University, Jinhua 321004, Zhejiang
⁴ Development Center of Science and Technology, Ministry of Agriculture and Rural Affairs, Beijing 100176

Received:2025-07-24 Accepted:2025-09-22 Published:2026-01-16 Online:2026-01-22

摘要/Abstract

摘要：

【目的】 转基因作物的田间快速筛查是生物安全监管的重要环节。针对当前转基因检测方法存在的设备依赖性强、操作复杂等技术瓶颈，以转基因大豆DBN9004为研究对象，开发基于重组酶聚合酶扩增（RPA）与劈裂型DNAzyme（MNAzyme）联用的闭管式快速检测技术，以实现转基因成分的快速、高灵敏、高特异性检测，为产业化安全监管提供可靠的技术支持。【方法】 以转基因大豆DBN9004及其受体Jack为试验材料。首先通过生物信息学方法筛选转化体特异性序列作为检测靶标，构建重组质粒9004P作为标准检测模板。采用非对称RPA反应体系，通过调控正向和反向引物浓度比例，在高效扩增目标序列的同时，大量生成激活MNAzyme所需的单链DNA（ssDNA）产物。在此基础上，建立双模输出系统（实时荧光监测模式/终点荧光成像模式）。系统优化关键反应参数：包括温度（35—60 ℃）、探针浓度（125—1 000 nmol·L^-1）、RPA引物比例（10 000﹕10 000 nmol·L^-1—10 000﹕31.25 nmol·L^-1）等。采用梯度稀释模板（8×10^-1—8×10⁵ copies/μL）评估方法灵敏度。通过10种转基因作物（GTS40-3-2、ZH10-6等）对方法进行特异性验证。最后采集13份田间样本进行实际样品检测，并与qPCR方法进行比较。【结果】 灵敏度方面，该方法在45 min内可稳定检出8 copies/反应的靶标DNA；方法重复性与再现性较好，相对标准偏差（RSD）分别为4.44%和5.75%；特异性试验显示，仅DBN9004产生显著荧光信号，与其他转基因品系无交叉反应；实际样品检测中，13份田间样本的检测结果与qPCR结果完全一致。【结论】 成功建立了RPA-MNAzyme联用的转基因大豆DBN9004快速检测技术。该方法通过非对称RPA与MNAzyme级联放大相结合，实现双重特异性识别与信号放大；开发闭管式检测体系，有效避免气溶胶污染；建立的双模输出系统可同时满足实验室与田间筛查需求。

关键词: 转基因作物, 重组酶聚合酶扩增, 劈裂型核酶, 等温扩增, 现场检测

Abstract:

【Objective】 Rapid on-site screening of genetically modified (GM) crops is crucial for effective biosafety regulation. To overcome the limitations of current detection methods, such as equipment dependency and operational complexity, this study developed a closed-tube detection system by integrating recombinase polymerase amplification (RPA) with split DNAzyme (MNAzyme). The system enables rapid, sensitive, and on-site detection of the GM soybean event DBN9004, supporting regulatory compliance and industrial safety management. 【Method】 Using GM soybean DBN9004 and its non-GM counterpart Jack as experimental materials, we firstly identified event-specific sequences for target detection through bioinformatics analysis. Then a recombinant plasmid (9004P) was constructed as a standard template. An asymmetric RPA system was designed to efficiently amplify the target sequence while generating abundant single-stranded DNA (ssDNA) products for MNAzyme activation. Critical reaction parameters were systematically optimized, including reaction temperature (35-60 ℃), probe concentration (125-1 000 nmol·L^-1), and RPA primer ratios (10 000﹕10 000 nmol·L^-1-10 000﹕31.25 nmol·L^-1). Sensitivity assessment was evaluated using gradient-diluted plasmids (8×10^-1-8×10⁵ copies/μL), while specificity evaluation was verified against ten GM crop lines (GTS40-3-2, ZH10-6, etc.). Field samples (n=13) were tested and compared with qPCR results. 【Result】 The method demonstrated exceptional sensitivity (8 copies/reaction), good repeatability (RSD=4.44%) and reproducibility (RSD=5.75%), absolute specificity for DBN9004 with no cross-reactivity against ten prevalent GM soybean varieties. Field testing demonstrated perfect concordance (100%) with qPCR results (n=13). 【Conclusion】 This study implemented an asymmetric RPA strategy to efficiently generate target-specific ssDNA amplicons. The resulting ssDNA products demonstrate specific binding affinity for pre-engineered split DNAzyme subunits (A/B), triggering their activation and subsequent continuous cleavage of fluorophore-quencher labeled substrate probes. Leveraging this molecular mechanism, we established a novel RPA-MNAzyme integrated platform for rapid and reliable detection of genetically modified soybean event DBN9004. By combining asymmetric RPA with MNAzyme cascade amplification, the method achieves dual-specificity recognition and signal enhancement. The closed-tube design prevents aerosol contamination, while the dual-mode output system accommodates both laboratory and on-site screening needs.

Key words: genetically modified crops, recombinase polymerase amplification, MNAzyme, isothermal amplification, on-site detection

符丽锦, 陈冠玮, 肖功, 汪小福, 彭城, 陈笑芸, 徐俊锋, 陈子言, 杨蕾. 基于DNAzyme信号放大的转基因大豆DBN9004快速检测方法[J]. 中国农业科学, 2026, 59(2): 239-249.

FU LiJin, CHEN GuanWei, XIAO Gong, WANG XiaoFu, PENG Cheng, CHEN XiaoYun, XU JunFeng, CHEN ZiYan, YANG Lei. A Rapid Detection Method for Genetically Modified Soybean Dbn9004 Based on Dnazyme Signal Amplification[J]. Scientia Agricultura Sinica, 2026, 59(2): 239-249.

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名称Name	序列Sequence (5′-3′)	产物大小Product size (bp)
DBN9004-F	CGGGACTTGATGAAAGAAGAG	254
FDBN9004-RR	GGTTTCGCTCATGTGTTGAG	254
9004-F4	ATGGCCGTATCCGCAATGTGTTATTAAGTT	108
9004-R3	AGAGCGTTGCATTTACGCTCCATAAACGTG
9004-M A1	ATTACGGGGTCAACAACGAGATGAATACTT
9004-M B1	ATCTGACGGAGGCTAGCTCAGAGTTGTAA
9004-M A2	CATTACGGGGTCAACAACGAGATGAATACTT
9004-M B2	ATCTGACGGAGGCTAGCTCAGAGTTGTAAA
9004-M A3	TCATTACGGGGTCAACAACGAGATGAATACTT
9004-M B3	ATCTGACGGAGGCTAGCTCAGAGTTGTAAAC
MNAzyme 探针 MNAzyme probe	(FAM)-CCACCACGAGTATTCATC/rG//rU/CCGTCAGACGTGGTGG-(BHQ1)
DBN9004-qF	AACGCGGCCG CTCTAG	165
S40031.4-qR	ATTATGTGGAAAGAAATGACCGAAA	165
DBN9004-qP	(FAM)-CGCCGGGTCCCGTTTAAACTATCAGT-(BHQ1)

分析方法 Analytical method	靶标 Target	检测限 LOD	耗时 Time (min)	参考文献Reference
多重PCR Multiplex PCR	特异性转化体Event-specific crops	0.1% (0.005 ng)	80	[25]
实时荧光定量PCR Real-time PCR	CP4-EPSPS	10 copies	90	[26]
环介导等温扩增 Loop-mediated isothermal amplification, LAMP	35S CP4-EPSPS, pat	0.50%	60	[27]
LAMP联合DNAzyme的试纸条方法 Multiplex LAMP-DNAzyme-LF	DP 305423 × GTS 40-3-2	0.1%	120	[28]
G-四链体-DNAzyme G-quadruplex DNAzyme	35S	5 nmol·L^-1	130	[29]
实时RPA Real-time recombinase polymerase amplification (RPA)	CP4-EPSPS, Cry1Ab/Ac	100 copies	20	[30]
RPA试纸条法 RPA-LFD	35S, NOS	50-100 copies	30	[31]
基于Cas12a检测法 Cas12a	SHZD32-1	0.3 fmol·L^-1, 3 fmol·L^-1	60	[32]
RPA联合Cas12a法 RPA-Cas12a	CP4-EPSPS/ Cry1Ab-Ac	45 copies	45	[33]
RPA联合Cas12a的试纸条法 RPA-Cas12a-FS	35S, NOS	10 copies	45	[34]
酶联免疫吸附测定法 ELISA	Cry1	15-30 ng·mL^-1	270	[35]
RPA联合MNAzyme方法 RPA-MNAzyme	特异性转化体DBN9004	8 copies	45	本研究This study

基于DNAzyme信号放大的转基因大豆DBN9004快速检测方法

A Rapid Detection Method for Genetically Modified Soybean Dbn9004 Based on Dnazyme Signal Amplification

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