Optimalizace kvalitativní a semikvantitivní detekce geneticky modifikovaných plodin pomocí PCR

Abstract

For qualitative and semi-quantitative detection of genetically modified crops we selected the detection of the frequently used promoter 35S CaMV. To optimise the method we used two commercially available genotypes of maize from the company Monsanto (USA), i.e. the transgenic hybrid Bt-maize line MON810 and a genetically non-modified control (isogenic line to MON810). We tested the pri­mers and PCR programmes described by Greiner et al. (2005) and Hernandéz et al. (2005). When applying PCR methods of detection of Bt-maize the first step was to optimise the protocol for the detection of the maize genome and detection of the specific sites of genetically modified MON810 maize. For detection of the maize genome we selected the primers IVR1-F and IVR1-R (invertase gene) which verify the presence of the maize genome by a 226 bp product. For qualitative detection of the insert of Bt-maize MON810 the primer pairs VW01/VW03 (Greiner et al., 2005) and BT03/BT04 (Hernandéz et al., 2005) were used to detect the 35S CaMV promoter. Products of the size 178 bp and 280 bp, respectively, verify its presence. Based on the results of qualitative PCR we selected the primers VW01/VW03 for semi-quantitative detection of the amount of DNA of Bt-maize. For semi-quantitative PCR we have chosen sampling of the amplification product in the 30th cycle of the PCR reaction. In the genetically unmodified control a detection limit of 1% of admixture of Bt-maize was determined when using semi-quantitative PCR. The same primers as for semi-quantitative PCR were also used for multiplex PCR but with half the concentration of primers for standard PCR. This protocol however will have to be further optimised. The presented results introduce PCR methods for qualitative and semi-quantitative detection of DNA of the genetically modified Bt-maize MON810 which can also be used for other GM crops containing the 35S CaMV promoter. It could be suitable to use these methods for the qualitative detection and/or for screening analyses of the detection of successfulness of transformation experiments.