Оптимизација и валидација на Real-Time PCR метод за анализа на генетски модифицирана храна

Abstract

The application of genetic engineering leads to changes in the genetic material of organisms by inserting a gene that carries information about a certain trait that is not naturally present in that organism. In this way, genetically modified organisms (GMOs) are designed with the desired characteristics. Despite the significant commercial value, consumers are still skeptical about the risks to the environment and health safety. In recent decades the number of genetically modified crops has increased rapidly, so their accurate detection is important for mandatory labelling and risk assessment. According to Regulations 1830/2003 and 1829/2003 of the European Union, the labelling of food and feed is mandatory if it contains a genetic modification in a concentration greater than 0.9%. Currently, the real-time PCR method is the „gold standard" for the analysis of genetically modified foods. Extraction of high quality DNA is a critical step for accurate and efficient DNA amplification. This doctoral dissertation describes a modification using the RNase A enzyme to an already optimized sodium dodecyl sulphate (SDS) protocol for the extraction of high quality DNA. This DNA is suitable for screening of genetic modification. The purpose of this dissertation was to develop, optimize and validate a screening method for the 35S promoter and Tnos terminator in raw and processed foods. During the research were optizmied the concentration and ratio of primers and TaqMan probe, the concentration of DNA template and of universal master mix. The lowest DNA concentration at which both genetic elements are detected is 0.1 ng/μL. The limit of detection (LOD) is 0.01% GMO. The method is accredited and is in the range of official methods of the Food Institute at Faculty of veterinary medicine – Skopje, used for routine analysis.