A novel isothermal detection method for the universal element of genetically modified soybean


Due to the increasingly heated debate on the potential threat of genetically modified (GM) crops to human health and environment, regulations and laws relevant to GM crops have been issued in many countries and regions to strictly restrict their cultivation and application. Therefore, fast and accurate method to realized on-site detection of GM crops is greatly demanded. In this work, a novel isothermal amplification method termed denaturation bubble-mediated strand exchange amplification (SEA) was proposed first time to detect GM crops. Fluorescence assay based on SEA could accurately distinguished GM and non-GM soybean by detecting agrobacterium tumefaciens nopaline synthase (NOS) terminator, which was widely incorporated in GM crops. Moreover, this feasible and specific method could detect NOS terminator from as low as 200 pg/μL total genomic DNA of GM soybean. In addition, in the actual sample detection, the result of colorimetric assay based on SEA results could be directly observed by the naked eyes within 58 min. Compared with the traditional methods based on PCR, which normally required complex equipment, skilled technicians and long operation time, this simple, fast and precise method is more desirable for the on-site GM crops detection.

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Data availability

We make sure that all data and materials support our published claims and comply with field standards.



Genetically modified


Nopaline synthase


Food and Drug Administration


United States Environmental Protection Agency


United States Department of Agriculture


Polymerase chain reaction


Strand exchange amplification


Loop-mediated isothermal amplification


Rolling-circle amplification


Recombinase polymerase amplification


Deoxyribonucleic acid


High performance liquid chromatography


National Center for Biotechnology Information


Deoxy-ribonucleoside triphosphates


No template control


Polyacrylamide gel electrophoresis


Threshold time


  1. Carvalho FP (2017) Pesticides, environment, and food safety. Food Energy Secur 6:48–60. https://doi.org/10.1002/fes3.108

    Article  Google Scholar 

  2. Deng J, Li Y, Shi W, Liu R, Ma C, Shi C (2020) Primer design strategy for denaturation bubble-mediated strand exchange amplification. Anal Biochem 593:113593. https://doi.org/10.1016/j.ab.2020.113593

    CAS  Article  PubMed  Google Scholar 

  3. Dona A, Arvanitoyannis IS (2009) Health risks of genetically modified foods. Crit Rev Food Sci 49:164–175. https://doi.org/10.1080/10408390701855993

    CAS  Article  Google Scholar 

  4. Faragová N, Gottwaldová K, Faragó J (2011) Effect of transgenic alfalfa plants with introduced gene for alfalfa mosaic virus coat protein on rhizosphere microbial community composition and physiological profile. Biologia 66:768. https://doi.org/10.2478/s11756-w011-0082-6

    Article  Google Scholar 

  5. Fraiture M, Herman P, Taverniers I, De Loose M, Deforce D, Roosens NH (2015) Current and new approaches in GMO detection: challenges and solutions. Biomed Res Int 2015. https://doi.org/10.1155/2015/392872

  6. Hrbek V, Krtkova V, Rubert J, Chmelarova H, Demnerova K, Ovesna J, Hajslova J (2017) Metabolomic strategies based on high-resolution mass spectrometry as a tool for recognition of GMO (MON 89788 variety) and non-GMO soybean: a critical assessment of two complementary methods. Food Anal Method 10:3723–3737. https://doi.org/10.1007/s12161-017-0929-8

    Article  Google Scholar 

  7. Kamle S, Ali S (2013) Genetically modified crops: detection strategies and biosafety issues. Gene 522:123–132. https://doi.org/10.1016/j.gene.2013.03.107

    CAS  Article  PubMed  Google Scholar 

  8. Kolm C, Mach RL, Krska R, Brunner K (2015) A rapid DNA lateral flow test for the detection of transgenic maize by isothermal amplification of the 35S promoter. Anal Methods-Uk 7:129–134. https://doi.org/10.1039/C4AY01997K

    CAS  Article  Google Scholar 

  9. Kou J, Tang Q, Zhang X (2015) Agricultural GMO safety administration in China. J Integr Agric 14:2157–2165. https://doi.org/10.1016/S2095-3119(15)61109-1

    Article  Google Scholar 

  10. Li R, Shi J, Liu B, Zhang D, Zhao X, Yang L (2018) International collaborative ring trial of four gene-specific loop-mediated isothermal amplification assays in GMO analysis. Food Control 84:278–283. https://doi.org/10.1016/j.foodcont.2017.08.012

    CAS  Article  Google Scholar 

  11. Li R, Shi J, Liu B, Wang C, Zhang D, Zhao X, Yang L (2019) Inter-laboratory validation of visual loop-mediated isothermal amplification assays for GM contents screening. Food Chem 274:659–663. https://doi.org/10.1016/j.foodchem.2018.07.010

    CAS  Article  PubMed  Google Scholar 

  12. Liu C, Shi C, Li M, Wang M, Ma C, Wang Z (2019a) Rapid and simple detection of viable foodborne pathogen Staphylococcus aureus. Front Chem 7:124. https://doi.org/10.3389/fchem.2019.00124

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. Liu R, Wang X, Wang X, Shi Y, Shi C, Wang W, Ma C (2019b) A simple isothermal nucleic acid amplification method for the effective on-site identification for adulteration of pork source in mutton. Food Control 98:297–302. https://doi.org/10.1016/j.foodcont.2018.11.040

    CAS  Article  Google Scholar 

  14. Liu S, Wei M, Liu R, Kuang S, Shi C, Ma C (2019c) Lab in a Pasteur pipette: low-cost, rapid and visual detection of Bacillus cereu using denaturation bubble-mediated strand exchange amplification. Anal Chim Acta 1080:162–169. https://doi.org/10.1016/j.aca.2019.07.011

    CAS  Article  PubMed  Google Scholar 

  15. Lizardi PM, Huang X, Zhu Z, Bray-Ward P, Thomas DC, Ward DC (1998) Mutation detection and single-molecule counting using isothermal rolling-circle amplification. Nat Genet 19:225. https://doi.org/10.1038/898

    CAS  Article  PubMed  Google Scholar 

  16. Long LK et al (2019) Rapid visual detection of four specific transgenic events in GM soybean using loop-mediated isothermal amplification method. Russ J Plant Phys l+66:646–655. https://doi.org/10.1134/S1021443719040071

    Article  Google Scholar 

  17. Ma C, Wang F, Wang X, Han L, Jing H, Zhang H, Shi C (2017) A novel method to control carryover contamination in isothermal nucleic acid amplification. Chem Commun 53:10696–10699. https://doi.org/10.1039/C7CC06469A

    CAS  Article  Google Scholar 

  18. McHughen A, Smyth S (2008) US regulatory system for genetically modified [genetically modified organism (GMO), rDNA or transgenic] crop cultivars. Plant Biotechnol J 6:2–12. https://doi.org/10.1111/j.1467-7652.2007.00300.x

    Article  PubMed  Google Scholar 

  19. Meric B, Kerman K, Marrazza G, Palchetti I, Mascini M, Ozsoz M (2004) Disposable genosensor, a new tool for the detection of NOS-terminator, a genetic element present in GMOs. Food Control 15:621–626. https://doi.org/10.1016/j.foodcont.2003.10.004

    CAS  Article  Google Scholar 

  20. Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, Hase T (2000) Loop-mediated isothermal amplification of DNA. Nucleic Acids Res 28:e63. https://doi.org/10.1093/nar/28.12.e63

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. Paternò A et al (2018) In-house validation and comparison of two wheat (Triticum aestivum) taxon-specific real-time PCR methods for GMO quantification supported by droplet digital PCR. Food Anal Method 11:1281–1290. https://doi.org/10.1007/s12161-017-1097-6

    Article  Google Scholar 

  22. Piepenburg O, Williams CH, Stemple DL, Armes NA (2006) DNA detection using recombination proteins. PLoS Biol 4:e204. https://doi.org/10.1371/journal.pbio.0040204

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. Shao N et al (2017) Visual detection of multiple genetically modified organisms in a capillary array. Lab Chip 17:521–529. https://doi.org/10.1039/C6LC01330A

    CAS  Article  PubMed  Google Scholar 

  24. Shi C, Shang F, Zhou M, Zhang P, Wang Y, Ma C (2016) Triggered isothermal PCR by denaturation bubble-mediated strand exchange amplification. Chem Commun 52:11551–11554. https://doi.org/10.1039/C6CC05906F

    CAS  Article  Google Scholar 

  25. Smyth SJ (2017) Genetically modified crops, regulatory delays, and international trade. Food Energy Secur 6:78–86. https://doi.org/10.1002/fes3.100

    Article  Google Scholar 

  26. Ulianas A, Heng LY, Lau H, Ishak Z, Ling TL (2014) Single-step and reagentless analysis of genetically modified soybean DNA with an electrochemical DNA biosensor. Anal Methods-Uk 6:6369–6374. https://doi.org/10.1039/C4AY00881B

    CAS  Article  Google Scholar 

  27. Wang K, Li X, Zhang J, Chen H, Zhang Z, Yu G (2010) Natural introgression from cultivated soybean (Glycine max) into wild soybean (Glycine soja) with the implications for origin of populations of semi-wild type and for biosafety of wild species in China. Genet Resour Crop Ev 57:747–761. https://doi.org/10.1007/s10722-009-9513-4

    Article  Google Scholar 

  28. Wang F, Feng J, Ye S, Huang H, Zhang X (2018) Development of a multiplex fluorescence quantitative PCR for detection of genetically modified organisms. Biologia 73:21–29. https://doi.org/10.2478/s11756-018-0004-y

    CAS  Article  Google Scholar 

  29. Zhang M, Wang X, Han L, Niu S, Shi C, Ma C (2018) Rapid detection of foodborne pathogen Listeria monocytogenes by strand exchange amplification. Anal Biochem 545:38–42. https://doi.org/10.1016/j.ab.2018.01.013

    CAS  Article  PubMed  Google Scholar 

  30. Zhou D et al (2016) Detection of bar transgenic sugarcane with a rapid and visual loop-mediated isothermal amplification assay. Front Plant Sci 7:279. https://doi.org/10.3389/fpls.2016.00279

    Article  PubMed  PubMed Central  Google Scholar 

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The authors sincerely thank Qingdao Grain and Oils Quality Inspection Center for GM bean samples. This work was supported by National Natural Science Foundation of China (No.31670868 and No.21675094), and Shandong Province Natural Science Fund Major Basic Research Project (ZR2017ZC0123).


This work was funded by National Natural Science Foundation of China (No.31670868 and No.21675094), and Shandong Province Natural Science Fund Major Basic Research Project (ZR2017ZC0123).

Author information




Yongxiang Liu and Yang Li performed the experiments; Yongxiang Liu, Yang Li and Mengzhe Li analyzed the data; Cuiping Ma and Chao Shi designed the study; Hongyuan Sun and Qingguo Huo provided the GM bean samples; Yang Li and Yongxiang Liu wrote the manuscript; and all authors contributed to the writing of the paper, had primary responsibility for the final content, and read and approved the final manuscript.

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Correspondence to Chao Shi.

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Liu, Y., Li, Y., Li, M. et al. A novel isothermal detection method for the universal element of genetically modified soybean. Biologia (2020). https://doi.org/10.2478/s11756-020-00541-8

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  • Isothermal nucleic acid amplification
  • SEA
  • Colorimetric detection
  • GM soybean
  • On-site detection