Samson, Maria Cristina
The Application of Molecular Biotechnology in implementing Regulatory Normatives: the case of GMOs [Tesi di dottorato]
Università degli Studi di Parma. Dipartimento di Scienze Ambientali, 2010-03-30T06:34:48Z

Biotechnology has been widely adopted in agriculture but this has been also the focus of many controversies. Questions have arisen regarding its impact on food and environmental safety. In Europe, EFSA (European Food Safety Authority) has primary responsibility in the food safety ensuring systems. Food safety assessment for the products obtained with or containing GMOs (Genetically Modified Organisms), requires evaluation of the safety of: the newly added DNA (Deoxyribonucleic Acid); the safety of the newly introduced gene product and the overall safety issues for new varieties crops that are potential toxicity of the newly introduced protein(s), potential changes in allergenicity, changes in nutrient composition, and unintended effects giving rise to allergenicity or toxicity. An issue that will gain importance in the near future is that of post-marketing surveillance of the foods derived from GM crops. As more GMOs enter the market worldwide, the monitoring is being preferred for obvious reasons such as determination of seed purity, verification of non-GMO status of agricultural crops and liability problems and to fulfill GMO labelling provisions. Numerous GMO analytical methods have been developed to reliably determine the presence and/or amount of GMO in agricultural commodities, in raw agricultural materials and in processed and refined ingredients. However, these methods need to be constantly updated because of the increasing use of newly developed traits or GMOs with stacked genes. The detection of GMOs relies on the detection of transgenic DNA or the transgenic protein. The methodologies for transgenic DNA detection are mainly based on the use of PCRbased (Polymerase Chain Reaction-based) techniques in particular, RTPCR (realtime PCR) is the most effective method for GMO quantification. The study discusses the development of multiplex real-time PCR methodologies to an easy applicable GMO analysis in model processed foods. The first chapter describes the development of duplex qRTPCR (quantitative real-time PCR) method for simultaneous detection and quantification of RRS (Roundup® Ready Soybean) line GTS 40-3-2 (Glyphosate Tolerant Soybean 40-3-2) targeting the junction region within the CP4-EPSPS / t-NOS (5- enolpyruvylshikimate-3-phosphate synthase/ Terminator nopaline synthase). The study is focused on the evaluation of the performance per se in determining and quantifying GM event in processed foods. This technique uses the fluorescent dye labelled TaqMan® MGB-NFQ (Minor Groove Binder - Non Fluorescent Quencher) chemistry that is specifically designed to help improve the performance of hybridization-based systems for genetic analysis. This method has the advantage of performing the amplification of the target taxon lectin gene (Le1) and the constructspecific genes of a GM soybean sequenced in the same tube. The second chapter focuses on the development of simultaneous amplification of two GM lines. This methodology will become practical especially in the determination of newly developed GM lines harboring stacked genes. The performance of the system has been evaluated using different endogenous control genes as well as the applicability of the developed multiplex methodology using SYBR® GreenER™ technique in the hope of developing a quantitative approach more handy than the classical real-time TaqMan® technology.

diritti: © Maria Cristina Samson, 2010
Marmiroli, Nelson
Gulli, Mariolina
BIO/13


Tesi di dottorato. | Lingua: Inglese. | Paese: | BID: TD13017914