Begüm TERZİ AKSOY, Özlem ATEŞ SÖNMEZOĞLU

İşlenmiş gıdalar için GDO tespitinde modifiye DNA izolasyon yöntemlerinin karşılaştırılması

İşlenmiş gıda numunelerindeki yüksek oranda bozulmuş DNA içeriği, GDO'ların tespitinde verimi sınırlamaktadır. Genel olarak, literatürde transgenik bitki tohumlarından veya ham maddelerden geleneksel DNA izolasyon teknikleri mevcuttur, ancak işlenmiş gıda örneklerinden DNA izolasyon teknikleri ile ilgili çalışmalar sınırlıdır. Ayrıca, birçok işlenmiş gıda ürünü, çok sayıda karmaşık bitki veya hayvan kaynaklarından elde edilen genomik DNA içerir. Bu çalışma bisküvi, kek, kraker, mısır cipsi ve un gibi işlenmiş gıdaların yüksek kaliteli DNA izolasyonu için bazı faydalı modifikasyonlar önermektedir. Bu amaçla, işlenmiş gıdaları incelemek için GDO analizinin ilk adımı olan gıda örneklerinden yüksek moleküler ağırlıklı ve kaliteli DNA elde etmek için izolasyon protokolleri araştırılmıştır. Elde edilen DNA örneklerinden beklenen uzunluktaki hedef organizmanın gen bölgesini kontrol etmek ve bunları DNA izolasyon verimleriyle karşılaştırmak için soya fasulyesi ve mısıra özgü primerler ile PCR tespiti yapılmıştır. Test edilen dört izolasyon yöntemi arasında, değiştirilmiş Wizard-CTAB yöntemi, test edilen gıda ürünlerinin çoğunda daha iyi sonuçlar vermiştir. Sonuçlar, modifiye Wizard-CTAB'nin mısır ve soya fasulyesine özgü genler ve GDO tespiti ile ilgili çalışmalar için farklı gıda ürünlerinde seçim için kullanılabileceğini göstermiştir.

Anahtar Kelimeler:

DNA izolasyonu, , genetiği değiştirilmiş organizma (GDO), lektin, nükleik asit eksraksiyonu, PCR tespiti

Comparison of Modified DNA Isolation Methods for the Detection of GMO in Processed Foods

The highly degraded DNA content in processed food samples results in limited efficiency in detecting GMOs. Generally, conventional DNA isolation techniques from transgenic plant seeds or raw materials are available in the literature, but studies on DNA isolation techniques from processed food samples are more limited. Also, many processed food products contain genomic DNA from numerous complex plants or animal sources. This study proposed some beneficial modifications for high-quality DNA isolation of processed foods such as biscuits, cakes, crackers, corn chips, and flours. For this purpose, isolation protocols were investigated to obtain high molecular weight and quality DNA from food samples, the first step in GMO analysis to study processed foods. PCR detection was performed with soybean and corn-specific primers to control the gene region of the target organism of expected length from the obtained DNA samples and compare them with DNA isolation yields. Among the four isolation methods tested, the modified Wizard-CTAB method showed better results in most of the tested food products. Results showed that the modified Wizard-CTAB could be used for selection can be made in different food products for studies on corn and soybean specific genes and GMO detection.

Keywords:

DNA isolation, genetically modified food (GMO), lectin, nucleic acid extraction, PCR detection,

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1. ISAAA, ISAAA Brief 55-2019: Executive Summary Biotech Crops Drive Socio-Economic Development and Sustainable Environment in the New Frontier. 2011. In: https://www.isaaa.org/resources/publications/briefs/55/executivesummary/default.asp.
2. Gryson, N., Effect of food processing on plant DNA degradation and PCR-based GMO analysis: a review. Analytical and Bioanalytical Chemistry, 2010. 396(6): p. 2003-2022.
3. Linnhoff, S., et al., An examination of millennials’ attitudes toward genetically modified organism (GMO) foods: is it Franken-food or super-food? International Journal of Agricultural Resources, Governance and Ecology, 2017. 13(4): p. 371.
4. Sönmezoğlu, Ö. and H. Keskin, Determination of genetically modified corn and soy in processed food products. Journal of Applied Biology & Biotechnology, 2015. (3): p. 32-37.
5. Arun, Ö.Ö., The effect of heat processing on PCR detection of genetically modified soy in bakery products. Journal of Food and Health Science, 2016. p. 130-139.
6. Pacheco Coello, R., et al. Comparison of three DNA extraction methods for the detection and quantification of GMO in Ecuadorian manufactured food. BMC Research Notes, 2017. 10(1): p. 758.
7. Saadedin, S., M. Abbas and A. Suleiman, Detection of CaMV-35S Promoter and NOS Terminator in Genetically Modified Tomato Seed in Iraqi Markets. Iraqi journal of biotechnology, 2019. 18(2).
8. Matthes, N., et al. Validation of a modified CTAB method for DNA extraction from protein-rich maize feedstuffs. Journal of Consumer Protection and Food Safety, 2020. 15(4): p. 331-340.
9. Ashrafi-Dehkordi, E., S.M. Mazloomi, and F. Hemmati, A comparison of DNA extraction methods and PCR-based detection of GMO in textured soy protein. Journal of Consumer Protection and Food Safety, 2021. 16(1): p. 51-57.
10. Tung Nguyen, C., et al. Comparison of DNA Extraction Efficiencies Using Various Methods for the Detection of Genetically Modified Organisms (GMOs). International Food Research Journal, 2009. 16: p. 21-30.
11. Doyle, J.J. and J.L. Doyle, Isolation of Plant DNA From Fresh Tissue. Focus (Madison), 1990. 12: p. 13-15.
12. Wilfinger, W.W., K. Mackey, and P. Chomczynski, Assessing the Quantity; Purity and Integrity of RNA and DNA following Nucleic acid purification. DNA sequencing II optimising preparation and cleanup, 2006. p. 291-312.
13. Vollenhofer, S., et al. Genetically Modified Organisms in FoodScreening and Specific Detection by Polymerase Chain Reaction. Journal of Agricultural and Food Chemistry, 1999. 47(12): p. 5038-5043.
14. Pauli, U., et al. Extraction and Amplification of DNA From 55 Foodstuffs. Mitteilungen aus Lebensmitteluntersuchung und Hygiene, 2000. 91: p. 491-501.
15. Cardarelli, P., et al. Detection of GMO in food products in Brazil: the INCQS experience. Food Control, 2005. 16(10): p. 859-866.
16. Lipp, M., et al. Validation of a method based on polymerase chain reaction for the detection of genetically modified organisms in various processed foodstuffs. European Food Research and Technology, 2001. 212(4): p. 497-504.
17. Ondřej, M.. Testing of Genetically Modified Organisms in Food, F.E., Ahmed, Editors. 2004, Biol Plant, 48(4): p. 516-516.
18. Laffont, J.L., et al. Testing for adventitious presence of transgenic material in conventional seed or grain lots using quantitative laboratory methods: statistical procedures and their implementation. Seed Science Research, 2005. 15(3): p. 197-204.
19. Shokere, L.A., M.J. Holden, and G. Ronald Jenkins, Comparison of fluorometric and spectrophotometric DNA quantification for real-time quantitative PCR of degraded DNA. Food Control, 2009. 20(4): p. 391-401.
20. Bauer, T., The effect of processing parameters on DNA degradation in food. European Food Research and Technology, 2003. 217(4): p. 338-343.
21. Gryson, N., K. Dewettinck, and K. Messens, Detection of Genetically Modified Soy in Doughs and Cookies. Cereal Chemistry Journal, 2007. 84(2): p. 109-115.
22. Sambrook, J., and D. Russell, Molecular cloning: a laboratory manual, 3rd ed. 2001. Cold Spring Harbor Laboratory Press, New York.
23. Ateş Sönmezoğlu, Ö., and B. Terzi, Characterization of some bread wheat genotypes using molecular markers for drought tolerance. Physiology and Molecular Biology of Plants, 2018. 24(1): p. 159-166.
24. Greiner, R., U. Konietzny, and A.L.C.H. Villavicencio, Qualitative and quantitative detection of genetically modified maize and soy in processed foods sold commercially in Brazil by PCR-based methods. Food Control, 2005. 16(8): p. 753-759.
25. Turkec, A., et al. DNA extraction techniques compared for accurate detection of genetically modified organisms (GMOs) in maize food and feed products. Journal of Food Science and Technology, 2015. 52(8): p. 5164-5171.
26. DiBernardo, G., et al. Comparative Evaluation of Different DNA Extraction Procedures from Food Samples. Biotechnology Progress, 2007. 23(2): p. 297-301.
27. Abdel-Latif, A., and G. Osman, Comparison of three genomic DNA extraction methods to obtain high DNA quality from maize. Plant Methods, 2017. 13(1): p. 1.
28. Pervaiz, Z.H., et al. Methodology A modified method for high-quality DNA extraction for molecular analysis in cereal plants. Genetics and Molecular Research, 2011. 10(3): p. 1669-1673.
29. Querci, M., et al. From sampling to quantification: developments and harmonisation of procedures for GMO testing in the European Union. Collection of Biosafety Reviews, 2007. 3: p. 8-14.
30. Özgen Arun, Ö., F., Yılmaz, and K. Muratoğlu, PCR detection of genetically modified maize and soy in mildly and highly processed foods. Food Control, 2013. 32(2): p. 525-531.
31. Anklam, E., et al. Analytical methods for detection and determination of genetically modified organisms in agricultural crops and plant-derived food products. European Food Research and Technology, 2002. 214(1): p. 3-26.
32. Rastegar, H., et al. Investigation of transgenic elements in genetically modified maize germ (Zea mays) and maize germ oil distributed in local market by qualitative PCR method. Human, Health and Halal Metrics, 2021. 1(2): p.64-70.
33. Jia, R., et al. Microbiologically influenced corrosion and current mitigation strategies: A state of the art review. International Biodeterioration & Biodegradation, 2019. 137: p. 42-58.
34. Aboul-Maaty, N.A.F., and H.A.S. Oraby, Extraction of high-quality genomic DNA from different plant orders applying a modified CTAB-based method. Bull Natl Res Cent, 2019. 43(1): p. 25.
35. Regulation (TR). Regulation of the Turkish Republic, Ministry of Food, Agriculture and Animal breeding on; Genetically Modified organisms and their products. 2010. Official journal 27671
36. Avsar, B., et al. Identification and quantitation of genetically modified (GM) ingredients in maize, rice, soybean and wheat-containing retail foods and feeds in Turkey. Journal of Food Science and Technology, 2020. 57(2): p.787-793.