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DNA purity and Real Time PCR kinetics : influence of the inhibitory effect on GMO quantitation

  • Janssen, E. , Debode, F. & Berben, G. (2003). DNA purity and Real Time PCR kinetics : influence of the inhibitory effect on GMO quantitation. Poster in: 1st International Symposium on Recent Advances in Food Analysis, Prague, 05-07/11/2003.
Type Poster
Year 2003
Title DNA purity and Real Time PCR kinetics : influence of the inhibitory effect on GMO quantitation
Event name 1st International Symposium on Recent Advances in Food Analysis
Event location Prague
Label 95
Recnumber 95
Event date 05-07/11/2003
Type of poster scientifique, recherche
Endnote keywords RA-CRA-W 2003-2004 Di-Biologie moléculaire Do-Biologie moléculaire
Endnote Keywords GMO|PCR|
Abstract Quantitation of GMO's (or any other target) by real time PCR requires an in depth knowledge of the kinetics of genetic amplification reactions. The aim of the presented work is to characterize the quality of DNA extracts by means of their influence on PCR kinetics. The two main traits that are assessed are the amplification efficiency and the possible inhibitory effect on PCR of an extract. Therefore three DNA isolation methods were used on fifteen matrices that are currently analysed in routine and of which some were known to have shown a very strong inhibitory effect. Several dilutions of these extracts were analysed by real time PCR. The target was either a DNA segment peculiar to the extract or a segment of plasmid DNA (internal control) added in same amount to all tested dilutions (moreover absence of this target born by the plasmid had been checked for each extract before addition of the plasmid). The obtained results clearly showed at least two kinds of possible inhibition patterns. In the first one, inhibition arises through a delay in the formation of amplification curves (the signal appears later on during cycling) but the exponential phase remains parallel to that of an amplification curve free of inhibition. In the second case, the inhibition results in a loss of amplification efficiency during the exponential phase (generally in conjunction with a delay of the signal). The first situation suggests that inhibition probably happens only during the early cycles of the reaction, when the exponential phase is not even visible in real time. While for the second case the inhibitory effect apparently goes on during the all reaction. That is why it can be hypothesized that the inhibition of the amplification in these two cases is due to molecules acting in different mechanisms. The main consequence of this statement is that when using single extract concentrations for samples in real time PCR, possible inhibition will be undetected and cause a bias for the purpose of quantitation of GMO's. This research was performed within the framework of the project "Tracing and authentication of GMO's and derived products in the agro-food sectors" (Research contract ° CP/42/322) coordinated by Dr. W. MOENS and funded by the Belgian Federal Offices for Scientific, Technical and Cultural Affairs (OSTC). We acknowledge Cécile Ancion, Aline Marien, Gaëlle Antoine and Denis Roulez for their technical assistance.
Author address Janssen Eric, Quality Department of Agro-food Products, Walloon Agricultural Research Centre (CRA-W), Chaussée de Namur, 24, B-5030 Gembloux, janssen@cra.wallonie.be
Fichier 150027852995-janssen-2003.ppt
Caption 95-janssen-2003.ppt
Authors Janssen, E. , Debode, F. & Berben, G.