Quantitation of genetically modified maize MON810 in two reference systems gives evidence of limitations in use of the conversion factor concept
- Janssen, E. , Hulin, J. , Debode, F. & Berben, G. (2004). Quantitation of genetically modified maize MON810 in two reference systems gives evidence of limitations in use of the conversion factor concept. Poster in: Rapid Methods Europe 2004, Noorkwijk aan Zee - The Netherlands, 25-26/03/2004.
|Quantitation of genetically modified maize MON810 in two reference systems gives evidence of limitations in use of the conversion factor concept
|Rapid Methods Europe 2004
|Noorkwijk aan Zee - The Netherlands
|Type of poster
|Di-Biologie moléculaire Do-Céréales RA-CRA-W 2003-2004
|When quantifying GMO content by real time PCR, there are presently two main calibration systems. The first one is based on use of flours made of ground kernels and having a known percent of mass fraction of genetically modified (GM) material in flour. In this system quantitative results on samples are to be expressed as a mass ratio supposed to represent the amount of GM material within the all mass of the considered ingredient. In the second system, calibration is based on copy numbers of the targets, now generally obtained through use of plasmids in known amounts. With this latter reference system, the GM content of a sample is to be expressed as a ratio (in %) between the measured copy number of the GM marker target in the sample and the measured amount of haploid genome equivalents in the sample. Due to biological reasons linked to zygosity and ploidy of the several plant tissues (especially in kernels), both calibration systems will not necessarily come up with identical figures but are supposed to be linked (let say per event to keep it easy) through a conversion factor (Cf) also named Cv for coefficient value by Japanese teams [1, 2]. The main objective of this work is to compare the figures obtained with two different types of calibration system for the quantitation of the GM maize MON810 by real time PCR. The comparison will establish what is the conversion factor between the expression of GMO content in % of mass fractions (IRMM MON810 standards) and the % of GM copies obtained through plasmids (Nippon gene Multi Target Plasmid) as reference material. Two samples (called comparison samples CS1 and CS2 with known contents of respectively 1.3% and 0.7% in mass fraction obtained in ring trials) completely unrelated to the standards were quantified within the two different reference systems and then compared. The conversion factors were determined with the help of figures obtained in the two reference systems for the comparison samples but also on each calibration point of the IRMM standard. Each of the reference flours and also the comparison samples were submitted to DNA extraction with the CTAB method. Real-time PCR was performed on an ABI Prism® 7000 according to conditions that had been optimised  and validated . The derived conversion factors were respectively of 0.60 and 0.62 for CS1 and CS2. Similar values ranging between 0.55 and 0.74 were obtained for each calibration point of the IRMM standards. Although results in this experiment are consistent they don't met the figures published by Japanese teams [1, 2] for single kernels (Cf of about 0.38 and 0.42). However the Japanese teams never determined the Cf on the IRMM standards. The origin of this discrepancy might be that grinding conditions of the IRMM material may have affected extractability of the different tissues of the maize kernels in a different way as the one performed in Japan. Noteworthy too is the fact that a technical document joined to IRMM standards  gives a measure of the number of MON810 DNA relative to total maize DNA in haploid genome equivalents for each IRMM MON810 standard. Conversion factors calculated from these data are grossly comparable to our results. Considering the several different conversion factors obtained with MON810 maize, we here give evidence that at least on maize, the concept of conversion factor should be handled with great caution if the aim is to convert copy number ratios in mass fractions. For the sake of harmonization of results between laboratories, it seems much more appropriate to express results in terms of copy number of a well selected GM marker per haploid genome equivalent as this is basically what real time PCR is able to measure. 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 Science Policy. Acknowledgements : We thank Dr. Philippe Corbisier (IRMM) for his collaboration.  Kuribara et al. (2002). J. AOAC Int. 85 (5)1077-1089.  Shindo et al. (2002). J. AOAC Int. 85 (5)1119-1126.  Trapmann et al. (2001) Doc. EUR 20111 EN. ISBN 92-894-1978-4.
|Janssen, E., Hulin, J., Debode, F., Berben, G.