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Loop-Mediated Isothermal Amplification (LAMP) Method for Rapid Detection of Rf1 Event in Transgenic Rapeseed (Brassica napus L.)

Received: 1 April 2022     Accepted: 18 April 2022     Published: 7 May 2022
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Abstract

Rapeseed (Brassica napus L.) is one of the most studied crops for genetic engineering. Up to now, at least 11 GM rapeseed varieties have been approved for commercial production. During this study, a simple and rapid LAMP method was established for detection of Rf1 event in transgenic rapeseed. Position 143-353 in the right border junction sequence of B. napus transgenic line Rf1 (accession number: EU090199.1) was chosen as the target region for primer design and LAMP detection. Reaction mixture at the volume of 25 μL contained 12.5 μL 2×buffer mix, 1.28 μM FIP, 1.28 μM BIP, 0.16 μM F3, 0.16 μM B3, 0.64 μM LF, 480 U/mL Bst DNA polymerase, 2.0 μL DNA template and 4.6 μL ddH2O. SYBR Green I was added to the tube lid. After incubation at 60°C for 60 min, the reaction was terminated by heating at 80°C for 10 min. Then the tube was centrifuged for 60 s at 8,000 r/min to mix dye with reaction mixture. Results indicate that our LAMP assay is highly specific and sensitive (9.2×103 copies/μL, 0.001%). This LAMP assay for Rf1 event in rapeseed, which is simple, time effective, specific, sensitive and result visible without expensive instruments, is suitable for application in wide fields.

Published in Journal of Plant Sciences (Volume 10, Issue 3)
DOI 10.11648/j.jps.20221003.11
Page(s) 86-90
Creative Commons

This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium or format, provided the original work is properly cited.

Copyright

Copyright © The Author(s), 2022. Published by Science Publishing Group

Keywords

Rf1 Event, Loop-Mediated Isothermal Amplification, Genetically Modified Organism, Sensitivity, Specificity

References
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[4] Sundby C, Chow WS, Anderson JM. Effects on photosystem II function, photoinhibition, and plant performance of the spontaneous mutation of serine-264 in the photosystem II reaction center D1 protein in triazine-resistant Brassica napus L. Plant physiology, 1993, 103 (1): 105-113.
[5] Zhou XY, Wu YY, Peng Q, et al. Indoor Propagation of Transgenic Glyphosate-tolerant Rapeseed (Brassica napus L.) and Transfer Breeding for Herbicide Tolerance. Agricultural Biotechnology (2164-4993), 2021, 10 (1): 1-5. 5p.
[6] Wu Y, Wu G, Xiao L, et al. Event-specific qualitative and quantitative PCR detection methods for transgenic rapeseed hybrids MS1×RF1 and MS1×RF2. Journal of agricultural and food chemistry, 2007, 55 (21): 8380-8389.
[7] Regulation No. 258/97 of the European Parliament and of the Council of 27 January 1997 concerning novel foods and novel food ingredients. Off. J. Eur. Communities: Legis. 1997, 43: 1-7.
[8] Matsuoka, T. GMO labeling and detection methods in Japan. APEC-JIRCAS Joint Symposium and Workshop on Agricltural Biotechnology, 2001.
[9] Li Q, Fang J, Liu X, et al. Loop-mediated isothermal amplification (LAMP) method for rapid detection of cry1Ab gene in transgenic rice (Oryza sativa L.). European Food Research and Technology, 2013, 236 (4): 589-598.
[10] Misawa Y, Saito R, Moriya K, et al. Application of loop-mediated isothermal amplification technique to rapid and direct detection of methicillin-resistant Staphylococcus aureus (MRSA) in blood cultures. Journal of Infection and Chemotherapy, 2007, 13 (3): 134-140.
[11] Garg N, Ahmad FJ, Kar S. Recent advances in loop-mediated isothermal amplification (LAMP) for rapid and efficient detection of pathogens. Current Research in Microbial Sciences, 2022: 100120.
[12] Kapalamula TF, Thapa J, Akapelwa ML, et al. Development of a loop-mediated isothermal amplification (LAMP) method for specific detection of Mycobacterium bovis. PLoS Neglected Tropical Diseases, 2021, 15 (1): e0008996.
[13] Yu Y, Li R, Ma Z, et al. Development and evaluation of a novel loop mediated isothermal amplification coupled with TaqMan probe assay for detection of genetically modified organism with NOS terminator. Food Chemistry, 2021, 356: 129684.
[14] Sorochynskyi BV. Detection of genetically modified plants using LAMP (loop-mediated amplification) technologies. Plant varieties studying and protection, 2021, 17 (1): 51-59.
[15] Huang X, Zhu SF, Gao HW, et al. Protocol of the real-time PCR method for detecting genetically modified plants and their derived products. SN/T 1204-2016. General Administration of Quality Supervision, Inspection and Quarantine of the People's Republic of China. 2016-6-28.
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  • APA Style

    Zhang Jihong, Wang Jiaying, Zhao Lei, Huang Suwen, Chen Xianfeng. (2022). Loop-Mediated Isothermal Amplification (LAMP) Method for Rapid Detection of Rf1 Event in Transgenic Rapeseed (Brassica napus L.). Journal of Plant Sciences, 10(3), 86-90. https://doi.org/10.11648/j.jps.20221003.11

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    ACS Style

    Zhang Jihong; Wang Jiaying; Zhao Lei; Huang Suwen; Chen Xianfeng. Loop-Mediated Isothermal Amplification (LAMP) Method for Rapid Detection of Rf1 Event in Transgenic Rapeseed (Brassica napus L.). J. Plant Sci. 2022, 10(3), 86-90. doi: 10.11648/j.jps.20221003.11

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    AMA Style

    Zhang Jihong, Wang Jiaying, Zhao Lei, Huang Suwen, Chen Xianfeng. Loop-Mediated Isothermal Amplification (LAMP) Method for Rapid Detection of Rf1 Event in Transgenic Rapeseed (Brassica napus L.). J Plant Sci. 2022;10(3):86-90. doi: 10.11648/j.jps.20221003.11

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  • @article{10.11648/j.jps.20221003.11,
      author = {Zhang Jihong and Wang Jiaying and Zhao Lei and Huang Suwen and Chen Xianfeng},
      title = {Loop-Mediated Isothermal Amplification (LAMP) Method for Rapid Detection of Rf1 Event in Transgenic Rapeseed (Brassica napus L.)},
      journal = {Journal of Plant Sciences},
      volume = {10},
      number = {3},
      pages = {86-90},
      doi = {10.11648/j.jps.20221003.11},
      url = {https://doi.org/10.11648/j.jps.20221003.11},
      eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.jps.20221003.11},
      abstract = {Rapeseed (Brassica napus L.) is one of the most studied crops for genetic engineering. Up to now, at least 11 GM rapeseed varieties have been approved for commercial production. During this study, a simple and rapid LAMP method was established for detection of Rf1 event in transgenic rapeseed. Position 143-353 in the right border junction sequence of B. napus transgenic line Rf1 (accession number: EU090199.1) was chosen as the target region for primer design and LAMP detection. Reaction mixture at the volume of 25 μL contained 12.5 μL 2×buffer mix, 1.28 μM FIP, 1.28 μM BIP, 0.16 μM F3, 0.16 μM B3, 0.64 μM LF, 480 U/mL Bst DNA polymerase, 2.0 μL DNA template and 4.6 μL ddH2O. SYBR Green I was added to the tube lid. After incubation at 60°C for 60 min, the reaction was terminated by heating at 80°C for 10 min. Then the tube was centrifuged for 60 s at 8,000 r/min to mix dye with reaction mixture. Results indicate that our LAMP assay is highly specific and sensitive (9.2×103 copies/μL, 0.001%). This LAMP assay for Rf1 event in rapeseed, which is simple, time effective, specific, sensitive and result visible without expensive instruments, is suitable for application in wide fields.},
     year = {2022}
    }
    

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  • TY  - JOUR
    T1  - Loop-Mediated Isothermal Amplification (LAMP) Method for Rapid Detection of Rf1 Event in Transgenic Rapeseed (Brassica napus L.)
    AU  - Zhang Jihong
    AU  - Wang Jiaying
    AU  - Zhao Lei
    AU  - Huang Suwen
    AU  - Chen Xianfeng
    Y1  - 2022/05/07
    PY  - 2022
    N1  - https://doi.org/10.11648/j.jps.20221003.11
    DO  - 10.11648/j.jps.20221003.11
    T2  - Journal of Plant Sciences
    JF  - Journal of Plant Sciences
    JO  - Journal of Plant Sciences
    SP  - 86
    EP  - 90
    PB  - Science Publishing Group
    SN  - 2331-0731
    UR  - https://doi.org/10.11648/j.jps.20221003.11
    AB  - Rapeseed (Brassica napus L.) is one of the most studied crops for genetic engineering. Up to now, at least 11 GM rapeseed varieties have been approved for commercial production. During this study, a simple and rapid LAMP method was established for detection of Rf1 event in transgenic rapeseed. Position 143-353 in the right border junction sequence of B. napus transgenic line Rf1 (accession number: EU090199.1) was chosen as the target region for primer design and LAMP detection. Reaction mixture at the volume of 25 μL contained 12.5 μL 2×buffer mix, 1.28 μM FIP, 1.28 μM BIP, 0.16 μM F3, 0.16 μM B3, 0.64 μM LF, 480 U/mL Bst DNA polymerase, 2.0 μL DNA template and 4.6 μL ddH2O. SYBR Green I was added to the tube lid. After incubation at 60°C for 60 min, the reaction was terminated by heating at 80°C for 10 min. Then the tube was centrifuged for 60 s at 8,000 r/min to mix dye with reaction mixture. Results indicate that our LAMP assay is highly specific and sensitive (9.2×103 copies/μL, 0.001%). This LAMP assay for Rf1 event in rapeseed, which is simple, time effective, specific, sensitive and result visible without expensive instruments, is suitable for application in wide fields.
    VL  - 10
    IS  - 3
    ER  - 

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Author Information
  • Technical Center, Ningbo Customs, Ningbo, China

  • Technical Center, Ningbo Customs, Ningbo, China

  • Ningbo Institute of Inspection and Quarantine Science and Technology, Ningbo, China

  • Technical Center, Ningbo Customs, Ningbo, China

  • Technical Center, Ningbo Customs, Ningbo, China

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