Abstract
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.
Similar content being viewed by others
Data availability
We make sure that all data and materials support our published claims and comply with field standards.
Abbreviations
- GM:
-
Genetically modified
- NOS:
-
Nopaline synthase
- FDA:
-
Food and Drug Administration
- US EPA:
-
United States Environmental Protection Agency
- USDA:
-
United States Department of Agriculture
- PCR:
-
Polymerase chain reaction
- SEA:
-
Strand exchange amplification
- LAMP:
-
Loop-mediated isothermal amplification
- RCA:
-
Rolling-circle amplification
- RPA:
-
Recombinase polymerase amplification
- DNA:
-
Deoxyribonucleic acid
- HPLC:
-
High performance liquid chromatography
- NCBI:
-
National Center for Biotechnology Information
- dNTPs:
-
Deoxy-ribonucleoside triphosphates
- NTC:
-
No template control
- PAGE:
-
Polyacrylamide gel electrophoresis
- Tt:
-
Threshold time
References
Carvalho FP (2017) Pesticides, environment, and food safety. Food Energy Secur 6:48–60. https://doi.org/10.1002/fes3.108
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
Dona A, Arvanitoyannis IS (2009) Health risks of genetically modified foods. Crit Rev Food Sci 49:164–175. https://doi.org/10.1080/10408390701855993
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
Smyth SJ (2017) Genetically modified crops, regulatory delays, and international trade. Food Energy Secur 6:78–86. https://doi.org/10.1002/fes3.100
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
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
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
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
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
Acknowledgments
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).
Funding
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
Authors and Affiliations
Contributions
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.
Corresponding author
Ethics declarations
Conflict of interest
There are no conflicts of interest to declare.
Consent to participate
All the authors are consent to participate to this work.
Consent for publication
All the authors are consent this manuscript publishing in Biologia.
Ethics approval
Not applicable.
Code availability
Not Applicable
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Rights and permissions
About this article
Cite this article
Liu, Y., Li, Y., Li, M. et al. A novel isothermal detection method for the universal element of genetically modified soybean. Biologia 75, 2395–2402 (2020). https://doi.org/10.2478/s11756-020-00541-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.2478/s11756-020-00541-8