Czech J. Genet. Plant Breed., 2002, 38(1):55-63 | DOI: 10.17221/6111-CJGPB

Comparison of different PCR-based protocols for detection of Roundup Ready soybean

J. Ovesná, L. Dedičová, J. Horáček, E. Sadilová, L. Kučera, L. Měsková
1 Research Institute of Crop Production - Division of Genetics and Plant Breeding, Prague-Ruzyně, Czech Republic
2 Agritec Ltd., Šumperk, Czech Republic

Genetically modified organisms have become a part of our environment and food chain. Roundup Ready soybean is at the moment the most frequent one that man can meet. National regulations require careful monitoring and detection of GMOs. We present in this investigation comparison of several protocols and individual steps, which are included in the whole detection procedure. Currently used CTAB based protocol is suitable for DNA isolation from the green plant tissue but also from the flour. Lectin coding sequence specific primers were suitable for soybean DNA detection unlike leu-tRNA gene specific primers. Only one of the tested CaMV sequence specific primer pairs did not amplify unspecific products under condition tested. NOS terminator specific primer pair was found suitable for routine screening. EPSPS gene was detected using two different protocols; the same primers did not identify other Roundup Ready GM crops.

Keywords: GMO; Roundup Ready soybean; PCR; detection

Published: March 31, 2002  Show citation

ACS AIP APA ASA Harvard Chicago Chicago Notes IEEE ISO690 MLA NLM Turabian Vancouver
Ovesná J, Dedičová L, Horáček J, Sadilová E, Kučera L, Měsková L. Comparison of different PCR-based protocols for detection of Roundup Ready soybean. Czech J. Genet. Plant Breed. 2002;38(1):55-63. doi: 10.17221/6111-CJGPB.
Download citation

References

  1. ANONYM (1986): Pesticide Fact Sheet: Glyphosate. Office of Pesticide Programs, U.S. Environmental Protection Agency, Washington, DC. EPA Publ. No. 540/FS-88-124.
  2. BARRY G.F., TAYLOR M.L., PADGETTE S.R., KOLACZ K.H., HALLAS L.E., DELLA-CIOPPA G., KISORE G.M. (1994): Cloning and Expression in Escherichia coli of the Glyphosate-to-Aminomethylphosphonic Acid Degrading Activity from Achromobacter sp. strain LBAA. Monsanto Technical Report MSLB13245, St. Louis.
  3. BERTHAU Y. (1999): Detection of GMO in raw and derived products: general guidelines for a normalized approach. In: Proc. 5th Int. Conf. Pests in Agriculture. Part I, Montpellier, France, 7-9 December: 15-16.
  4. CROON K.S. (1996): Petition for Determination of Nonregulated Status: Insect-protected Roundup Ready Corn Line MON 802. USDA Petition, No. 96-317-01P.
  5. DELLA-CIOOPA G., BAUER S.C., KLEIN B.K., SHAH D.M., F RALEY R.T., KISHORE G. (1986): Translocation of the precursor of 5-enolpyruvylshikimate-3-phosphate synthase into chloroplasts of higher plants in vitro. Proc. Natl. Acad. Sci. U.S.A., 83: 6873-6877. Go to original source... Go to PubMed...
  6. DELLA-CIOPPA G., BAUER S.C., TAYLOR M.T., ROCHESTER D.E., KLEIN B.K., SHAH D.M., FRALEY R.T., KISHORE G.M. (1987): Targeting a herbicide-resistant enzyme from Escherichia coli to chloroplasts of higher plants. Bio/Technol.. 5: 579-584. Go to original source...
  7. DUIJN G. van, BIERT R. van, BLEEKER-MARCELIS H., PEPPELMAN H., HESSING M. GRASSERBAUER M. (1999): Detection methods for genetically modified crops. KNOWLES M., KUIPER H.A., GENDRE F. (eds.): Detection methods for novel food derived from genetically modified organisms. Food Control. (Spec. Issue), 10: 375-378. Go to original source...
  8. DUKE S.O. (1996): Herbicide Resistant Crops. CRC Press, New York.
  9. GADANI F., BINDLER G., PIJNENBURG H., ROSSI L., ZUBER J. (2000): Current PCR methods for the detection, identification and quantification of genetically modified organisms (GMOs): a brief review. Beitr. Tabakfors. Int., 19: 85-96. Go to original source...
  10. GACHET E., MARTIN G.G., VIGNEAU F., MEYER G. (1998): Detection of genetically modified organisms (GMOs) by PCR: a brief review of methodologies available. Trends Food Sci. Technol., 9: 380-388. Go to original source...
  11. INIS M.A, GELFAND D.H., SNINSKY J.J., SNINSKY J.I. (1995): The PCR Strategies. Acad. Press, New York.
  12. JANKIEWICZ A., BROLL H., ZAGON J. (1999): An official method for the detection of genetically modified soybeans (German Food Act LMBG 35): a semi-quantitative study of sensitivity limits with glyphosate-tolerant soybeans (Roundup Ready) and insect-resistant Bt maize (Maximizer). Eur. Food Res. Technol., 209: 77-82. Go to original source...
  13. KINOUCHI M., KANAYA S., IKEMURA T., KUDO Y. (2000): Detection of tRNA Based on the Cloverleaf Secondary Structure. In: DUNKER K., KONAGAYA A., MIYANO S., TAKAGI T. (eds.): Genome Informatics 2000. Univ. Acad. Press, Tokyo, 11: 301-302.
  14. KISHORE G., SHAH D. (1988): Amino acid biosynthesis inhibitors as herbicides. Annu. Rev. BioChem., 57: 627-663. Go to original source... Go to PubMed...
  15. LEVIN J.G., SPRINSON D.B. (1964): The enzymatic formation and isolation of 5-enolpyruvylshikimate-3-phosphate. J. Biol. Chem., 239: 1142-1150. Go to original source...
  16. LIPP M., ANKLAM E., BRODMANN P., PIETSCH K., PAUWELS J., GASSERBAUER M. (2001): Results of an interlaboratory assessment of a screening method of genetically modified organisms in soya beans and maize. In: KNOWLES M., KUIPER H.A., GENDRE F. (eds.): Detection methods for novel food derived from genetically modified organisms. Food Control. (Spec. Issue), 10: 379-383. Go to original source...
  17. LIPP M., BLUTH A., EYQUEM F., KRUSE L., SCHIMMEL H., EEDE G. van den, ANKLAM E. van den, EEDE G. (1999a): Validation of a method based on polymerase chain reaction for the detection of genetically modified organisms in various processed foodstuffs. Eur. Food Res. Technol., 212: 497-504. Go to original source...
  18. LIPP M., BRODMANN P., PIETSCH K., PAUWELS J., ANKLAM E. (1999b): UPAC collaborative trial study of a method to detect genetically modified soy beans and maize in dried powder. J. AOAC Int., 82: 923-928. Go to original source... Go to PubMed...
  19. LU F.C. (1995): A review of the acceptable daily intakes of pesticides assessed by the World Health Organization. Regul. Toxicol. Pharmacol., 21: 351-364. Go to original source... Go to PubMed...
  20. MARTYNKOVÁ R., OVESNÁ J., KUČERA L. (1997): Evaluation of wheat (Triticum aestivum L.) cultivars with standard Gli/Glu alleles using PCR and leaf tissue as a direct template. Czech J. Genet. Plant Breed., 33: 251-260.
  21. MEYER R., CHARDONNENS F., HUBNER P., LUTHY J. (1996): Polymerase Chain Reaction (PCR) in the quality and safety assurance of food detection of soya in processed meat product. Z. Lebensmittel Unters. Fors., 203: 339-344. Go to original source... Go to PubMed...
  22. MONSANTO (1997): Petition for determination of nonregulated status: Roundup Ready Corn Line GA21 (Submitted to United States Department of Agriculture), Petition No. 97-099-01P.
  23. MULLIS K.B., FALOORA F. (1987): Specific synthesis of DNA in vitro via a polymerase catalyzed chain reaction. Methods Enzymol., 155: 335-350. Go to original source... Go to PubMed...
  24. PASQUALONE A. (2000): Analytical methods for detecting genetically modified organisms in food: rules, theoretical basis and critical points. Industrie-Alimentari, 39: 444-451.
  25. PECCHIONI N., FACCIOLI P., MONETTI A., STANCA M., TERZI V. (1996): Molecular markers for genotype identification in small grain cereals. J. Genet. Breed., 50: 203-219.
  26. P IETSCH K., W AIBLINGER H., B RODMANN P., W URZ A. (1997): Screeningverfahren zur Identifizierung "genetisch verandered". Pfl. Lebensmit., Dtsch. Lebensmit. Rundschau, 93: 35-38.
  27. SAGHAI-MAROOF M.A., SOLIMAN K.M., JORGENSEN R.A., ALLARD R.W. (1984): Ribosomal DNA spacer-length polymorphism in barley. Mendelian inheritance, chromosomal location and population dynamics. Proc. Natl. Acad. Sci., 81: 8014-8018. Go to original source... Go to PubMed...
  28. SHAH D., HORSCH R., KLEE H., KISHOR G., WIINTER J., TUMER N., HIRONAKA C., SANDERS P., GASSER C., AYKENT S., SIEGEL N., ROGERS S., FRALEY R. (1986): Engineering herbicide tolerance in transgenic plants. Science, 233: 478-481. Go to original source... Go to PubMed...
  29. STEINRUCKEN H.C., AMRHEIN N. (1980): The herbicide glyphosate is a potent inhibitor of 5-enolpyruvyl-shikimate-3phosphate synthase. Biochem. Biophys. Res. Comm., 94: 1207-1212. Go to original source... Go to PubMed...
  30. STEINRUCKEN H.C., AMRHEIN N. (1984): 5-Enolpyruvylshikimate-3-phosphate synthase of Klebsiella pneumoniae: inhibition by glyphosate [N-(phosphonomethyl)-glycine]. Eur. J. Biochem., 143: 351-357. Go to original source... Go to PubMed...
  31. VOLLENHOFER S., BURG K., SCHMIDT J., KROATH H. (1999): Genetically modified organisms in food - screening and specific detection by polymerase chain reaction. J. Agr. Food Chem., 47: 5038-5043. Go to original source... Go to PubMed...
  32. WAUCHOPE R.D., BUTTLER T.M., HORNSBY A.G., AUGUSTIJN BECKERS P.W.M., BURT J.P. (1992): SCS/ARS/CES pesticide properties database for environmental decisionmaking. Rev. Environ. Contam. Toxicol., 123: 1-157. Go to original source... Go to PubMed...
  33. WOLF C., SCHERZINGER M., WURZ A., PAULI U., HUBNER P., LUTHY J. (2000): Detection of cauliflower mosaic virus by the polymerase chain reaction: testing of food components for false positive 35S promotor screening results. Eur. Food Res. Technol., 210: 367-372. Go to original source...

This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY NC 4.0), which permits non-comercial use, distribution, and reproduction in any medium, provided the original publication is properly cited. No use, distribution or reproduction is permitted which does not comply with these terms.