Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress

Giardini Naxos, Italy - 18/21 September, 2002

ISBN 88-900622-3-1

Poster Abstract - 2.23

MOLECULAR GENETICS METHODOLOGIES FOR DNA TRACEABILITY THROUGH THE AGRO-ALIMENTARY CHAIN

PEANO C., PALMIERI L., SAMSON C., PALOMBA A., MARMIROLI N.

Sez. Genetica e Biotecnologie Ambientali, Dip. Scienze Ambientali, Università di Parma

traceability, food, DNA extraction, Real Time PCR, PNA

The use of high quality raw material in food production is a prerequisite in order to achieve a genuine and secure product of adequate nutritional value. In the European Community traceability of origin, quality and authenticity of food products is becoming very important. It will therefore become necessary to develop appropriate techniques in order to correctly trace and label foods. In this context, and to avoid alimentary frauds, the need for efficient and reliable detection methods is clear.Up to now, DNA or ribonucleic acid (RNA) have been of minor interest in food analysis and there is therefore little knowledge about detection of nucleic acid (NA) in many processed foodstuffs. Although many DNA extraction protocols were available, they have rarely been compared in a comprehensive manner. The presence of DNA in foodstuffs which are, or contain, genetically modified organisms (GMO) is the basic requirement for labelling of GMO foods in the European Union, that’s why we chose to develop a model system of DNA traceability on food products containing known percentages of GMO deriving material.

In the first phase of the work, four different methods for DNA extraction where evaluated and compared each other. To evaluate the different methods we considered the quality and the quantity of DNA extracted from several standards, containing known percentages of GMO material, and from different food products. In this work we extracted DNA from standard flours containing different GMO material deriving from RoundupReady™ Soybean, from Maize Bt176, MON810 and GA21. The food products analysed in this study derived both from Soybean and Maize material and they where chosen considering also the mechanical, technological and chemical treatment they have been subjected. Degree of DNA degradation at various stages of food production was evaluated through the amplification of different DNA size fragments.

In the second phase of this work we used DNA extracted from the standards to sequence the transgenic constructs contained in: RoundupReady™ Soybean, Maize Bt176, MON810, GA21.Thanks to the sequences obtained in the second phase of the work we were able to design specific probes useful for GMO quantification with Real Time PCR, and also to design Peptide Nulcleic Acids (PNA) useful in the PNA-mediated PCR-Clamping.

In the third phase of the work the presence of GMOs was quantified by using Real Time PCR. PNA-mediated PCR-Clamping was used as a qualitative detection method and in particular as a practical and fast post-PCR control. This is based on Peptide Nucleic Acid (PNA) oligomers designed on the sequences of the transgenic constructs analysed. The selective blocking of PCR amplification due to these oligomers allows both the confirmation of the identity of the amplified band and a qualitative estimate of the GMO content.

The aim of this study is to support the identification of possible frauds and agro-food manipulations.