Investigating The GMO Existence in Chips and Breakfast Cereals Marketed in Turkey

Year 2021, , 375 - 385, 07.09.2021

Şebnem Mutlu , Osman Şimşek , Ömer Öksüz

https://doi.org/10.33462/jotaf.670008

Cited By: 1

Abstract

In this research, processed or low processed samples containing corn or corn products (corn semolina, flour, etc.) and soybean were randomly collected from the market, and 25 products in total (chips, nuts, cereals, flour) were analyzed for genetic modification using DNA based detection method, the polymerase chain reaction. First, homogenization of the samples was performed. Then DNA isolation was done by using Cetyl Trimethyl Ammonium Bromide (CTAB) and Roche High Pure DNA Isolation Kit. Since the Roche High Pure DNA Isolation Kit gave better results, the analysis was completed with this method. After DNA isolation, the detection of the Lectin gene, Zein gene, CaMV 35S Promoter and NOS Terminator regions was performed by conventional PCR. Zein gene determination was done for searching and proving corn presence and similarly, Lectin gene determination was done for searching and proving soybean presence in the samples by conventional PCR. GMO3/GMO4 and Zein3/Zein4 primer pairs were used for Lectin and Zein gene determination, respectively. The amplification of DNA was observed in agarose gel electrophoresis. Lectin or Zein genes were detected in 17 samples while these genes were not detected in 8 samples. Samples, in which Lectin or Zein gene was detected were scanned for 35S promoter or NOS terminator. 35S-3/35S-6 and tNOS2F/tNOS2R primer pairs were used for scanning 35S Promoter and NOS Terminator, respectively. To observe possible contamination in the mix sterilized deionized water was used and 0% Bt-11 and 0% GTS 40-3-2 were used as a negative control, 5% Bt-11 and 10% GTS-40-3-2 were used as a positive control. All of the 25 samples did not provide enough DNA with the required quality. This result was considered to be sourced by the applications (frying, extruding, pressing etc.) that samples had been exposed to during processes. Neither 35S Promotor nor NOS Terminator was determined from any of the samples.

Keywords

GMO, Convantional PCR, Lectin, Zein, 35S Promotor, NOS Terminator

Supporting Institution

NKU Bilimsel Araştırma Projeleri Koordinasyon Birimi

Project Number

NKUBAP.00.24.AR.13.13 .

Thanks

We thank to Namık Kemal University Scientific Research Projects Coordination Department (NKUBAP) for financial support. (Project Number NKUBAP.00.24.AR.13.13)

Investigating The GMO Existence in Chips and Breakfast Cereals Marketed in Turkey

Abstract

Bu çalışmada piyasada satılan mısır ya da mısır ürünleri (mısır irmiği, mısır unu vb.) veya soya içeren işlenmiş veya yarı işlenmiş ürünler rastgele seçilerek toplamda 25 adet üründe (cips, çerez, gevrek, kuruyemiş kaplaması, un) GDO varlığı DNA temelli bir tespit metodu olan konvansiyonel Polimeraz Zincir Reaksiyonu (PCR) ile araştırılmıştır. Bu amaçla homojenize edilen örneklerde CTAB (Cetyl Trimethyl Ammonium Bromide) yöntemi ve Roche High Pure DNA Isolation Kit ile DNA izole edilerek çoğaltılması denenmiştir. DNA Isolation Kit kullanılarak DNA izole edilmesi yöntemi daha başarılı olduğundan işlemler bu yöntemle tamamlanmıştır. DNA izolasyonunun ardından Lektin geni, Zein geni, 35S Promotör ve NOS Terminatör bölgelerinin tespiti konvansiyonel PCR ile yapılmıştır. Zein geni tespiti örneklerdeki mısır varlığını, Lektin geni tespiti ise örneklerdeki soya varlığını araştırmak ve kanıtlamak amacıyla yapılmıştır. Zein ve Lektin genleri tespiti için sırasıyla GMO3/GMO4 ve Zein3/Zein4 primer çiftleri kullanılmıştır. Elde edilen DNA’ların amplifikasyonundan sonra DNA agaroz jel elektroforezi ile gözlemlenmiştir. Agaroz jel elektroforezi sonucu alınan görüntülerde 17 örnekte Lektin ya da Zein geni tespit edilirken 8 örnekte bu genler tespit edilememiştir. Lektin ya da Zein geni tespit edilen ürünlerin 35S Promotör ve NOS Terminatör taraması için çalışılmaya devam edilmiştir. Bunun için sırasıyla 35S-3/35S-6 ve tNOS2F/tNOS2R primer çiftleri kullanılmıştır. Mümkün kontaminasyonları gözlemlemek için sterilize su kullanılırken, negatif kontrol amacıyla 0 % Bt-11 ve 0% GTS 40-3-2, pozitif kontrol amacıyla ise 5% Bt-11 ve 10% GTS-40-3-2 kullanılmıştır. 25 örneğin hiçbirinde istenen kalitede ve yeterli miktarda DNA elde edilememesinin sebebinin işleme sırasında uygulanan prosesler (kızartma, kavurma, ekstrude etme, basınç vb.) olduğu düşünülmektedir. Hiçbir numunede 35S Promotör ve NOS Terminatör bölgesine rastlanmamıştır.

Keywords

GDO, Konvansiyonel PCR, Lektin geni, Zein geni, 35S Promotör, NOS Terminatör

Project Number

NKUBAP.00.24.AR.13.13 .

References

• Abdel-Latif, A., Osman, G. (2017). Comparison of three genomic DNA extraction methods to obtain high DNA quality from maize. Plant Methods, 13:1,1-9.
• Altındal, D., Altındal, N., Akgün, İ. (2017). Tritikale (X Triticosecale Wittmack) genotiplerinin ISSR-PCR yöntemi ile moleküler düzeyde tanımlanması. Journal of Tekirdag Agricultural Faculty, 14 (03): 19-26.
• Aydın, G. (2004). Detection and Quantification of Genetically Modified Maize Via Polymerase Chain Reaction. Msci. Thesis, Department of Biotechnology, METU, Ankara
• Ballari, R.V., Martin, A. (2013). Assessment of DNA degradation induced by thermal and UV radiation processing: Implications for quantification of genetically modified organisms. Food Chemistry, 141:2130-2136.
• Bauer, T., Hammes, W.P., Haase, N.U., Hertel, C. (2004). Effect of food components and processing parameters on DNA degradation in food. Environ. Biosafety Res., 3:215-223.
• Bergen, G. (2001). Final Report Contract NP/42/026.
• Bergerova, E., Hrnčírová, Z., Stankovská, M., Lopašovská, M., Siekel, P. (2010). Effect of thermal treatment on the amplification and quantification of transgenic and non-transgenic soybean and maize DNA. Food Anal. Methods, 3: 211–218.
• Brara, Z., Costa, J., Villa, C., Grazina, L., Bitam, A., Mafra, I. (2020). Surveying genetically modified maize in foods marketed in Algeria. Food Control, 109.
• Cankar, K., Stebih, D., Dreo, T., Zel, J., Gruden, K (2006). Critical points of DNA quantification by real-time PCR-effects of DNA extraction method and sample matrix on quantification of genetically modified organisms. BMC Biotechnol., 6:37–51.
• Chakraborty, S., Saha, A., Anathram, A.N. (2020). Comparison of DNA extraction methods for non‑marine molluscs: is modified CTAB DNA extraction method more efficient than DNA?. Protocols and Methods, 10:69.
• Chao, E. (2007). A Risk-Based Classification Scheme for Genetically Modified Foods:Establishing Levels of Concern to Guide Testing Requirements. Faculty of Graduate and Post Doctoral Studies. University of Ottawa Canada, 19-75.
• Chen, Y., Wang, Y., Ge, Y., Xu, B. (2005). Degradation of endogenous and exogenous genes of roundup-ready soybean during food processing. J. Agric. Food Chem., 53: 10239–10243.
• Çakar, T. (2010). Genetiği Değiştirilmiş Organizmalar ve Tüketici Hakları. Farklı Boyutlarıyla Genetiği Değiştirilmiş Organizmalar, Ankara, 75-84.
• Çakmak, A.I. (2010). Türkiye’ de Satışa Sunulan Bazı Gıda Ürünlerinde Genetik Modifiye Mısır Bileşenlerinin Aranması. [Detection of Genetically Modified Corn in Foods Sold in Turkey].Yüksek Lisans Tezi, İstanbul Teknik Üniversitesi, Fen Bilimleri Enstitüsü. İTÜ, İstanbul.
• Ekinci, M.B. (2008). İşlenmiş ve İşlenmemiş Bazı Mısır Ürünlerinde Genetik Modifikasyonun Tespiti. [Detection of Genetically Modified Organisms in Processed and Unprocessed Corn Foods]. Doktora Tezi, Fen Bilimleri Enstitüsü, Ankara Üniversitesi Gıda Mühendisliği Anabilim Dalı. Ankara.
• Fernandes, T.J.R., Costa, J., Placido, A., Villa, C., Grazina, L., Meira, L., Oliveira, M.B.P.P., Mafra, I. (2016). Genetically Modified Organism Analysis as Affected by DNA Degradation, Editor: R.R. WATSON and V.R. PREEDY, Genetically Modified Organisms in Food (P. 111-118), Academic Press, Portugal.
• Gachet, E.; Martin, G.G.; Vigneau, F.; Meyer, G. (1999). Detection Of Genetically Modified Organisms (Gmos) By PCR: A Brief Review Of Methodologies Available. Trends in Food Science and Technology, 9: 380-388.
• Gryson, N., Messens, K., Dewettinck, K. (2004). Evaluation and optimisation of five different extraction methods for soy DNA in chocolate and biscuits extraction of DNA as a first step in GMO analysis. J Sci Food Agric., 81:231–234.
• Gryson, N., Dewettinck, K., Messens, K. (2007). Detection of genetically modified soy in doughs and cookies. Cereal Chem., 84: 109–115.
• Guerra, F.X.M. (2005). Development of technique fort the quantification of DNA from genetically modified organisms in processed foods. Lehrsthul fur Allegement Lebensmittettechnologie. Techisque Universitat München. Retrieved from: https://mediatum.ub.tum.de/doc/603550/603550.pdf
• Güngören, A.V. (2012). Genetiği Değiştirilmiş Tarım Ürünlerinin Türkiye Açısından Değerlendirilmesi. [The Evaluation of Genetically Modified Agricultural Products in terms of Turkey]. Yüksek Lisans Tezi. Anakara Üniversitesi Fen Bilimleri Enstitüsü, Ankara.
• Gurakan, G.C., Aydın, G., Yılmaz, R. (2011). Qualitative detection of GM maize (Bt11) in food and feed sold commercially in Turkey by PCR based methods. Indian Journal of Biotechnology, 10: 143-146.
• Hemmer, W. (1997). Foods Derived From Genetically Modified Organisms And Detection Methods. Retrieved From: http://www.bats.ch/bats/publikationen/1997-2_gmo/gmo_food.pdf
• Horuz, S., Aysan, Y. (2018). Kabakgil tohumlarında karpuz bakteriyel fide yanıklığı ve meyve lekesi hastalığı etmeni Acidovorax citrulli’nin varlığının belirlenmesinde kullanılabilecek uygun yöntem(ler)in saptanması. Journal of Tekirdag Agricultural Faculty, 15 (03): 36-43.
• Hrncirova, Z., Bergerova, E., Siekel, P. (2008). Effects of technological treatment on DNA degradation in selected food matrices of plant origin. J. Food Nutr. Res., 47: 23–28.
• Jen Lu I.; Lin, C.; Ming, T. (2010). Establishment Of A System Based On Universal Multiplex-PCR For Screening Genetically Modified Crops. Pan Anal Bioanal Chem, 396: 2055–2064.
• Kay, S. and Paoletti, S. (2001). Sampling strategies for GMO detection and/or quantification. Rev 4.2, 23 Nov. 2001. EC. Directorate general JRC. IHCP (Institute for Healt and Consumer Protection). Food Products Unit: GMO: Food and Environment.
• Kaynar, P. (2010). Genetik Olarak Değiştirilmiş Organizmalar (GDO)’a Genel Bir Bakış. Türk Hijyen ve Deneysel Biyoloji Dergisi. 66: 177-185.
• Kıran, F.; Osmanağaoğlu, Ö. (2011). Gıdalarda Genetik Yapısı Değiştirilmiş Organizmaların (GDO) Belirlenmesi. Gıda, 36(5): 295-302.
• Kulaç, İ.; Ağırdil, Y.; Yakın, M. (2006). Sofralarımızdaki Tatlı Dert, GDO ve Halk Sağlığına Etkileri. Türk Biyokimya Dergisi., 31(3):151-155.
• Lipp, M. and Anklam, E. (2002). Validation of an immunoassay for the detection and quantification of roundop-ready soybean in food and feed fraction by the use of reference materials. European Commission Joint research Centre. Institute for Healt and Consumer Protection. Food Production and Consumer Goods Unit. 260p. Ispra, Italy.
• Mafra, I., Silva S.A., Moreira, E.J.M.O., Ferreira da Silva, C.S., Beatriz, M., Oliveira, P.P. (2008). Comparative study of DNA extraction methods for soybean derived food products. Food Control, 19:1183–1190.
• McBride, K.; Summerfelt, K. (1990). Improved Binary Vectors For Agrobacterium Mediated Plant Transformation. Plant Mol Biol, 14: 269–276.
• Meriç, S.; Çakır, Ö.; Turgut, K.N. and Arı, Ş. (2014). Detection Of Genetically Modified Maize And Soybean in Feed Samples. Gen. and Mol. Res. 2014; 13: 160-1168.
• Meyer, R. (1999). Development And Application Of DNA Analytical Methods For The Detection Of Gmos in Food. Food Control, 10: 391-399.
• Murray, S.R., Butler, R.C., Timmerman-Vaughan, G.M. (2009). Quantitative real-time PCR assays to detect DNA degradation in soy-based food products. J. Sci. Food Agric., 89: 1137–1144.
• Özmert Ergin, S. and Yaman, H. (2013). Genetiği Değiştirilmiş Gıdalar ve İnsan Sağlığı Üzerine Etkileri. Gümüşhane Üniversitesi Sağlık Bilimleri Dergisi, 2(2): 261-274.
• Pauli, U.; Liniger, M.; Zimmerman, A.; Schrott, M. (2000). Extraction and amplification of DNA from 55 foodstuffs. Mitt. Lebensm. Hyg. , 91: 491-500.
• Querci, M.; Van den Eede, G. and Jermini, M. (2002). The analysis of food samples for the presence of genetically modified organisms. European Commission. Joint Research Centre Manual. 2, 5-8.
• Rizzi, A., Panebianco, L., Giaccu, D., Sorlini, C., Daffonchio, D. (2003). Stability and recovery of maize DNA during food procecessing. Ital J Food Sci, 15:499–510.
• Sathe, S.K., Sharma, G.M. (2009). Effects of food processing on food allergens. Mol. Nutr. Food Res., 53: 970–978.
• Smith, D.S., Maxwell, P.W. (2007). Use of quantitative PCR to evaluate several methods for extracting DNA from corn flour and cornstarch. Food Control, 18:236–242.
• Stefanova, P., Taseva, M., Georgieva, T., Gotcheva, V., Angelov, A. (2013). A Modified CTAB Method for DNA Extraction from Soybean and Meat Products. Biotechnology & Biotechnological Equipment, 27:3, 3803-3810.
• Terry, C.F., Harris, N., Parkers, H.C. (2002). Detection of genetically modified crops and their derivatives: Critical steps in sample preparation and extraction. Journal of AOAC International, 85: 768–774.
• Tung-Nguyen, C.T., Son, R., Raha, A.R., Lai, O.M., Clemente Michael, W.V.L. (2009). Comparison of DNA extraction efficiencies using various methods for the detection of genetically modified organisms (GMOs). Int Food Research J. ,16:21–30.
• Turhan, A., Kafkas, S. (2013). Soya ve Mısırda Genetiği Değiştirilmiş Ürünlerin Belirlenmesi. Çanakkale University, Fen ve Mühendislik Bilimleri Dergisi, 29(2): 80-89.
• Turkec, A., Kazan, H., Karacanli, B., Lucas, S.J. (2015). DNA extraction techniques compared for accurate detection of genetically modified organisms (GMOs) in maize food and feed products. J Food Sci Technol, 52(8):5164–5171.
• Turkec, A., Lucas, S.J., Karlik, E. (2016). Monitoring the prevalence of genetically modified (GM) soybean in Turkish food and feed products. Food Control, 59: 766-772.
• Vijayakumar, K.R., Martin, A., Gowda, L.R., Prakash, V. (2009). Detection of genetically modified soya and maize: impact of heat processing. Food Chem., 117: 514–521.