Scientia Agricultura Sinica ›› 2026, Vol. 59 ›› Issue (2): 239-249.doi: 10.3864/j.issn.0578-1752.2026.02.002

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

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. 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 Online:2026-01-16 Published:2026-01-22
  • Contact: CHEN ZiYan, YANG Lei

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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

Table 1

The sequence list of the oligonucleotides in this study"

名称Name 序列Sequence (5′-3′) 产物大小Product size (bp)
DBN9004-F CGGGACTTGATGAAAGAAGAG 254
FDBN9004-RR GGTTTCGCTCATGTGTTGAG
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
DBN9004-qP (FAM)-CGCCGGGTCCCGTTTAAACTATCAGT-(BHQ1)

Fig. 1

The principle and workflow of RPA-MNAzyme combined detection method"

Fig. 2

Feasibility of RPA-MNAzyme combined detection method"

Fig. 3

Optimization of Reaction Performance of RPA-MNAzyme combined detection method A: Selection of optimal MNAzyme (9004-M A/B); B: Probe concentration optimization; C: Working temperature optimization; D: RPA primer ratio optimization. F: Fluorescence intensity of the corresponding conditions with target; F0: Blank fluorescence intensity of the corresponding conditions without target"

Fig. 4

Sensitivity assessment of RPA-MNAzyme combined detection method A: Real-time detection curves at various DBN9004 DNA concentrations; B: Standard curve established based on gradient dilution template; C: Bar charts and fluorescence images document RPA-MNAzyme combined detection method endpoint signals. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. The horizontal dashed line indicates the threshold fluorescence intensity. NTC: Non-template control. The same as below"

Fig. 5

Repeatability and reproducibility results of RPA combined MNAzyme assay"

Fig. 6

Specificity assessment of RPA-MNAzyme combined detection method"

Fig. 7

Robust performance of RPA combined with MNAzyme method in soybean seeds detection A: Bar charts and fluorescence images of RPA-MNAzyme combined detection method for soybean seed samples; B: Real-time detection curves of soybean seed samples using qPCR; C: Correlation between parallel measurements of soybean seeds using RPA-MNAzyme detection method (Fluorescence intensity, y-axis) and qPCR (Ct values, x-axis). NC: Negative control; PC: Positive control"

Table 2

Comparison of GM-related methods"

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
[1]
USLU T. Advantages, risks and legal perspectives of GMOs in 2020s. Plant Biotechnology Reports, 2021, 15(6): 741-751.

doi: 10.1007/s11816-021-00714-0
[2]
ROMEIS J, MEISSLE M, BIGLER F. Early-tier tests insufficient for GMO risk assessment-Reply. Nature Biotechnology, 2007, 25(1): 36-37.

doi: 10.1038/nbt0107-36
[3]
MARTINEZ-RIBAYA B, AREAL F J. Is there an opportunity for product differentiation between GM and non-GM soya-based products in Argentina? Food Control, 2020, 109: 106895.

doi: 10.1016/j.foodcont.2019.106895
[4]
AKIYAMA H, MAKIYAMA D, NAKAMURA K, SASAKI N, MINEGISHI Y, MANO J, KITTA K, OZEKI Y, TESHIMA R. A novel detection system for the genetically modified canola (Brassica rapa) line RT73. Analytical Chemistry, 2010, 82(23): 9909-9916.

doi: 10.1021/ac102434q pmid: 21049930
[5]
张月秋, 傅芳奇, 张华, 陈一帆, 王晨尧, 蒋红叶, 李亚辉, 廖一尘, 王丹, 孙宇, 等. 转基因耐除草剂大豆LP012-1 转化体实时荧光定量PCR检测方法的研究. 生物技术进展, 2025, 15(2): 276-286.
ZHANG Y Q, FU F Q, ZHANG H, CHEN Y F, WANG C Y, JIANG H Y, LI Y H, LIAO Y C, WANG D, SUN Y, et al. Research of detection method for genetically modified herbicide-tolerant soyabean LP012-1 transformant by quantitative real-time PCR. Current Biotechnology, 2025, 15(2): 276-286. (in Chinese)

doi: 10.19586/j.2095-2341.2024.0208
[6]
陆佳雨, 王沛然, 许学, 吴爽, 胡笑林, 李晨, 潘伟芹, 江雅婷, 汪秀峰. 转基因抗虫大豆转化体CAL16定性PCR检测方法. 生物安全学报(中英文), 2025, 34(2): 137-144.
LU J Y, WANG P R, XU X, WU S, HU X L, LI C, PAN W Q, JIANG Y T, WANG X F. Qualitative PCR detection method for transgenic insect-resistant soybean transformant CAL16. Journal of Biosafety, 2025, 34(2): 137-144. (in Chinese)
[7]
LAW J W, AB MUTALIB N S, CHAN K G, LEE L H. Rapid methods for the detection of foodborne bacterial pathogens: Principles, applications, advantages and limitations. Frontiers in Microbiology, 2014, 5: 770.
[8]
NOTOMI T, OKAYAMA H, MASUBUCHI H, YONEKAWA T, WATANABE K, AMINO N, HASE T. Loop-mediated isothermal amplification of DNA. Nucleic Acids Research, 2000, 28(12): e63.

doi: 10.1093/nar/28.12.e63 pmid: 10871386
[9]
REID M S, LE X C, ZHANG H Q. Exponential isothermal amplification of nucleic acids and assays for proteins, cells, small molecules, and enzyme activities: An EXPAR example. Angewandte Chemie, 2018, 57(37): 11856-11866.
[10]
WU X Y, CHEN S T, ZHANG Z X, ZHANG Y H, LI P M, CHEN X Y, LIU M M, LU Q, LI Z Y, WEI Z Y, et al. Development of recombinase polymerase amplification combined with lateral flow strips for rapid detection of cowpea mild mottle virus. The Plant Pathology Journal, 2023, 39(5): 486-493.

doi: 10.5423/PPJ.OA.02.2023.0033
[11]
KIM S H, LEE S Y, KIM U, OH S W. Diverse methods of reducing and confirming false-positive results of loop-mediated isothermal amplification assays: A review. Analytica Chimica Acta, 2023, 1280: 341693.

doi: 10.1016/j.aca.2023.341693
[12]
ZHAO Y X, CHEN F, LI Q, WANG L H, FAN C H. Isothermal amplification of nucleic acids. Chemical Reviews, 2015, 115(22): 12491-12545.

doi: 10.1021/acs.chemrev.5b00428 pmid: 26551336
[13]
AHMAD MUNAWAR M. Critical insight into recombinase polymerase amplification technology. Expert Review of Molecular Diagnostics, 2022, 22(7): 725-737.

doi: 10.1080/14737159.2022.2109964
[14]
DAHER R K, STEWART G, BOISSINOT M, BOUDREAU D K, BERGERON M G. Influence of sequence mismatches on the specificity of recombinase polymerase amplification technology. Molecular and Cellular Probes, 2015, 29(2): 116-121.

doi: 10.1016/j.mcp.2014.11.005 pmid: 25481659
[15]
CHEN J S, MA E B, HARRINGTON L B, DA COSTA M, TIAN X R, PALEFSKY J M, DOUDNA J A. CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science, 2018, 360(6387): 436-439.

doi: 10.1126/science.aar6245 pmid: 29449511
[16]
DING L, WANG X F, CHEN X Y, XU X L, WEI W, YANG L, JI Y, WU J, XU J F, PENG C. Development of a novel Cas13a/Cas12a- mediated ‘one-pot’ dual detection assay for genetically modified crops. Journal of Advanced Research, 2025, 72: 97-106.

doi: 10.1016/j.jare.2024.07.027
[17]
MUKAMA O, WU J H, LI Z Y, LIANG Q X, YI Z J, LU X W, LIU Y J, LIU Y M, HUSSAIN M, MAKAFE G G, et al. An ultrasensitive and specific point-of-care CRISPR/Cas12 based lateral flow biosensor for the rapid detection of nucleic acids. Biosensors & Bioelectronics, 2020, 159: 112143.

doi: 10.1016/j.bios.2020.112143
[18]
MOKANY E, BONE S M, YOUNG P E, DOAN T B, TODD A V. MNAzymes, a versatile new class of nucleic acid enzymes that can function as biosensors and molecular switches. Journal of the American Chemical Society, 2010, 132(3): 1051-1059.

doi: 10.1021/ja9076777 pmid: 20038095
[19]
YANG Y, SHI Y H, ZHANG X L, LI G L. MNAzyme catalyzed signal amplification-mediated lateral flow biosensor for portable and sensitive detection of mycotoxin in food samples. Analytical and Bioanalytical Chemistry, 2024, 416(4): 1057-1067.

doi: 10.1007/s00216-023-05096-6 pmid: 38117324
[20]
STEPHEN W, SANTORO G F J. A general purpose RNA-cleaving DNA enzyme. Proceedings of the National Academy of Sciences of the United States of America, 1997, 94(9): 4262-4266.
[21]
ZHOU Z X, BRENNAN J D, LI Y F. A multi-component all-DNA biosensing system controlled by a DNAzyme. Angewandte Chemie International Edition, 2020, 59(26): 10401-10405.
[22]
HANPANICH O, OYANAGI T, SHIMADA N, MARUYAMA A. Cationic copolymer-chaperoned DNAzyme sensor for microRNA detection. Biomaterials, 2019, 225: 119535.

doi: 10.1016/j.biomaterials.2019.119535
[23]
YANG L, CHEN G W, WU J, WEI W, PENG C, DING L, CHEN X Y, XU X L, WANG X F, XU J F. A PAM-free one-step asymmetric RPA and CRISPR/Cas12b combined assay (OAR-CRISPR) for rapid and ultrasensitive DNA detection. Analytical Chemistry, 2024, 96(14): 5471-5477.

doi: 10.1021/acs.analchem.3c05545 pmid: 38551977
[24]
GAO Y, TANG J, ZHOU Q, YU Z C, WU D, TANG D P. Excited-state intramolecular proton transfer-driven photon-gating for photoelectrochemical sensing of CO-releasing molecule-3. Analytical Chemistry, 2024, 96(12): 5014-5021.
[25]
SUH S M, KIM H J, SHIN M K, HONG S J, CHA J E, KIM H Y. Multiplex PCR detection method of genetically modified canola event (MON94100, LBFLFK, and NS-B50027-4) combined with capillary electrophoresis. Food Science and Biotechnology, 2024, 33(3): 637-643.

doi: 10.1007/s10068-023-01377-z
[26]
YANG L T, YANG Y, JIN W J, ZHANG X J, LI X Y, WU Y H, LI J, LI L. Development and interlaboratories validation of event-specific quantitative real-time PCR method for genetically modified rice G6H1 event. Journal of Agricultural and Food Chemistry, 2018, 66(30): 8179-8186.

doi: 10.1021/acs.jafc.8b01519 pmid: 29985602
[27]
TAKABATAKE R, KAGIYA Y, MINEGISHI Y, YEASMIN S, FUTO S, NOGUCHI A, KONDO K, MANO J, KITTA K. Development and evaluation of rapid screening detection methods for genetically modified crops using loop-mediated isothermal amplification. Food Chemistry, 2018, 252: 390-396.

doi: S0308-8146(17)31985-4 pmid: 29478558
[28]
CHENG N, SHANG Y, XU Y C, ZHANG L, LUO Y B, HUANG K L, XU W T. On-site detection of stacked genetically modified soybean based on event-specific TM-LAMP and a DNAzyme- lateral flow biosensor. Biosensors and Bioelectronics, 2017, 91: 408-416.

doi: 10.1016/j.bios.2016.12.066
[29]
JIANG X H, ZHANG H M, WU J, YANG X, SHAO J W, LU Y J, QIU B, LIN Z Y, CHEN G N. G-quadruplex DNA biosensor for sensitive visible detection of genetically modified food. Talanta, 2014, 128: 445-449.

doi: 10.1016/j.talanta.2014.05.002 pmid: 25059184
[30]
XU C, LI L, JIN W J, WAN Y S. Recombinase polymerase amplification (RPA) of CaMV-35S promoter and nos Terminator for rapid detection of genetically modified crops. International Journal of Molecular Sciences, 2014, 15(10): 18197-18205.

doi: 10.3390/ijms151018197
[31]
LIU H, WANG J B, LI P, BAI L, JIA J W, PAN A H, CUI W D, TANG X M. Rapid detection of P-35S and T-nos in genetically modified organisms by recombinase polymerase amplification combined with a lateral flow strip. Food Control, 2020, 107: 106775.

doi: 10.1016/j.foodcont.2019.106775
[32]
GE H R, WANG X F, XU J F, LIN H, ZHOU H Q, HAO T T, WU Y B, GUO Z Y. A CRISPR/Cas12a-mediated dual-mode electrochemical biosensor for polymerase chain reaction-free detection of genetically modified soybean. Analytical Chemistry, 2021, 93(44): 14885-14891.

doi: 10.1021/acs.analchem.1c04022 pmid: 34698496
[33]
WANG J B, HU X W, WANG Y, ZENG H J, LIU X F, LIU H. Rapid detection of genetically modified products based on CRISPR-Cas12a combined with recombinase polymerase amplification. Current Research in Food Science, 2022, 5: 2281-2286.

doi: 10.1016/j.crfs.2022.11.009 pmid: 36439643
[34]
LIU H, WANG J B, ZENG H J, LIU X F, JIANG W, WANG Y, OUYANG W B, TANG X M. RPA-Cas12a-FS: A frontline nucleic acid rapid detection system for food safety based on CRISPR-Cas12a combined with recombinase polymerase amplification. Food Chemistry, 2021, 334: 127608.

doi: 10.1016/j.foodchem.2020.127608
[35]
DONG S, ZHANG C Z, ZHANG X, LIU Y, ZHONG J F, XIE Y J, XU C X, DING Y, ZHANG L Q, LIU X J. Production and characterization of monoclonal antibody broadly recognizing Cry1 toxins by use of designed polypeptide as hapten. Analytical Chemistry, 2016, 88(14): 7023-7032.

doi: 10.1021/acs.analchem.6b00429 pmid: 27341419
[36]
WANG B L, JIAO Y L, LI X X, ZHENG F, LIANG H, SUN Z Y, GUO G. A universal method for directional cloning of PCR products based on asymmetric PCR. Biotechnology and Applied Biochemistry, 2009, 52(1): 41-44.

doi: 10.1042/BA20070210
[37]
KOBER C, NIESSNER R, SEIDEL M. Quantification of viable and non-viable Legionella spp. by heterogeneous asymmetric recombinase polymerase amplification (HaRPA) on a flow-based chemiluminescence microarray. Biosensors & Bioelectronics, 2018, 100: 49-55.

doi: 10.1016/j.bios.2017.08.053
[38]
ELSÄßER D, HO J, NIESSNER R, TIEHM A, SEIDEL M. Heterogeneous asymmetric recombinase polymerase amplification (HaRPA) for rapid hygiene control of large-volume water samples. Analytical Biochemistry, 2018, 546: 58-64.

doi: S0003-2697(18)30071-X pmid: 29412142
[39]
YANG L, CHEN G W, BO Y M, PENG C, YAN J T, CHEN X Y, XU X L, WEI W, FANG X X, WU J, et al. An ultra-sensitive quarantine pathogen on-site detection based on a one-pot asymmetric recombinase polymerase amplification and MNAzyme-assisted target recycling biosensor (OAR-MNA). Plant Biotechnology Journal, 2025, 23 (12), 5387-5396.

doi: 10.1111/pbi.v23.12
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