OPTIMIZATION OF REAL TIME PCR PARAMETERS BY CENTRAL COMPOSITE DESIGN IN GMO ANALYSIS OF CROP PLANT

Year 2022, Volume: 13 Issue: 26, 659 - 681, 27.12.2022

Özlem Akçay Kasapoğlu , Meryem Demircioğlu

https://doi.org/10.36543/kauiibfd.2022.028

Abstract

Toplam kalite yönetiminde, karşılaşılan problemleri bir daha ortaya çıkmayacak şekilde çözmek temel hedeftir. Deney tasarımı, her alanda geniş bir şekilde uygulanan istatistiksel bir teknik olup kalitenin geliştirilmesinde birçok katkıda bulunmaktadır. Bu çalışma, deney tasarımı tekniklerini kullanarak mısırda GDO (Genetiği Değiştirilmiş Organizma) gen bölgesinin aranması ve düzgün bir şekilde çoğaltılması için gerçek zamanlı PCR (Polimeraz Zincir Reaksiyonu) parametrelerinin çok yanıtlı optimizasyonu sağlanarak çabuk, güvenilir ve en düşük maliyetle GDO analizinin gerçekleştirilmesini amaçlamaktadır. Bu çalışmada birçok riske sahip olduğu düşünülen genetiği değiştirilmiş ürünlerin tespitinde rol oynayan ct değeri ve PCR ürün konsantrasyonuna etki eden faktörler ve bu faktörlerin seviyeleri tespit edilmiştir. Bu faktörlerin yanıt değerlerine olan etkileri Minitab.18 programında yüzey yanıt yöntemlerinden merkezi kompozit tasarım metodu ile belirlenmiş ve faktörler optimize edilmiştir. Optimum faktör seviyeleri kullanılarak doğrulama deneyleri gerçekleştirilerek oluşturulan model doğrulanmıştır.

Keywords

GDO, gerçek zamanlı PCR, merkezi kompozit tasarım

OPTIMIZATION OF REAL TIME PCR PARAMETERS BY CENTRAL COMPOSITE DESIGN IN GMO ANALYSIS OF CROP PLANT

Abstract

In total quality management, the main goal is to solve the problems encountered in a way that will not occur again. Experimental design is a statistical technique widely applied in every field and makes many contributions to the improvement of quality. This study aims to perform GMO (Genetically Modified Organism) analysis quickly, reliably and at the lowest cost by providing multi-response optimization of real-time PCR (Polimerase Chain Reaction) parameters to search and properly amplify the GMO gene region in maize using experimental design techniques. In this study, the ct value, which plays a role in the detection of genetically modified products, which are thought to have many risks, and the factors affecting the PCR product concentration and the levels of these factors were determined. The effects of these factors on the response values were determined by the central composite design method, one of the surface response methods, in Minitab.18 program and the factors were optimized. The model was validated by performing validation experiments using optimum factor levels.

Keywords

GDO, real time PCR, central composite design

References

• Abdel-Fattah, Y. R., & Gaballa, A. A. (2006). Synthesis of DNA Ladder by Polymerase Chain Reaction and Optimization of Yield Using Response Surface Methodology. Biotechnology, 166-172.
• Anonimous, (2013a). TS EN ISO 21571 Foodstuffs - Analysis Methods for the Determination of Genetically Modified Organisms and Derived Products - Nucleic Acid Extraction.
• Anonimous, (2013b). TS EN ISO 24276 Foods - Analysis Methods for Detection of Genetically Modified Organisms and Derived Derivatives - Recipes and General Rules.
• Anonimous, (2016). Event-specific method for the quantitation of Maize DAS-40278-9 using real- time PCR /EURL Metot.
• Anonimous, (2018). Eurofins GeneSpin Catalogue, Kit for isolation of high-Quality DNA from Food and Feed Samples, Cat. No. 522440605.
• Boleda, M. D., Briones, P., Farrés J., Tyfield, L., & Pi, R. (1996). Experimental Design: A Useful Tool for PCR Optimization. BioTechniques, 134-140.
• Camacho, J. L., Torres, E. M., Cadena, C., Prieto, J., Prieto, L. L., & Torregroza, D. A. (2013). The Use of Factorial Design, Image Analysis, and an Efficiency Calculation for Multiplex PCR Optimization. Journal of Clinical Laboratory Analysis, 249-252.
• Cobb, B. D., & M.Clarkson, J. (1994). A simple procedure for optimising the polymerase chain sreaction (PCR) using modified Taguchi methods. Nucleic Acids Research, s. 3801-3805.
• Nabi, N., Zellama, M. S., Hafsa, A. B., & Chaouachi, M. (2016). A new Multiplex PCR-based strategy to detect GM maize events with 2k fractional factorial plan in processed food. Journal of Research in Biological Sciences, 35-43.
• Niens, M., Spijker, G. T., Diepstra, A., & Meerman, G. J. (2005). A factorial experiment for optimizing the PCR conditions in routine genotyping. Biotechnol. Appl. Biochem., 157-162.
• Souza, H. J., Moyses, C. B., Pontes, F. J., Duarte, R. N., Silva, C. E., Alberto, F. L., & Silva, M. B. (2011). Molecular assay optimized by Taguchi experimental design method for venous thromboembolism investigation. Molecular and Cellular Probes, 231-237.
• Thanakiatkrai, P., & Welch, L. (2011). Using the Taguchi method for rapid quantitative PCR optimization with SYBR Green I. Int J Legal Med.
• Zhang, Q. Y., Zhou W.W., Zhou Y., Wang X. F., & Xu, J.F. (2021). Response Surface Methodology to Design a Selective Co Enrichment Broth of Escherichia coli, Salmonella Spp. and Staphylococcus aureus for Simultaneous Detection by Multiplex PCR, Microbiological Research, 167, 405-412.