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.
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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
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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).
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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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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
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DOI: https://doi.org/10.2478/s11756-020-00541-8