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VYHNANEK, T., HANACEK, P.: Optimisation of qualitative and semi-quantitative detection of genetically modifi ed crops by PCR. Acta univ. agric. et silvic. Mendel. Brun., 2009, LVII, No. 5, pp. 313–318 For qualitative and semi-quantitative detection of genetically modifi ed 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-modifi ed control (isogenic line to MON810). We tested the primers and PCR programmes described by Greiner et al. (2005) and Hernandez et al. (2005). When applying PCR methods of detection of Bt-maize the fi rst step was to optimise the protocol for the detection of the maize genome and detection of the specifi c sites of genetically modifi ed 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 (Hernandez 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 amplifi cation product in the 30th cycle of the PCR reaction. In the genetically unmodifi ed 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 semiquantitative detection of DNA of the genetically modifi ed 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. PCR, qualitative detection, semi-quantitative detection, multiplex PCR, transgenic crops, maize MON810 Along with the development of new transgenes and growing cultivation of genetically modifi ed plants (GMP) the requirements (by law) for their identifi cation, diff erentiation and marking are increasing too. The Zea mays L. line MON810 from Monsanto (YieldGard®) is one of the 18 GMO (genetically modifi ed organism) authorizations granted to date in the EU for placing onto the market. This GMO contains a genome integrated plant expression cassette comprised of the caulifl ower mosaic virus 35S promoter and hsp70 maize intron sequences driving expression of a synthetic cryIA(b) gene. The cryIA(b) gene codes for delta-endotoxin which binds to specifi c sites localized on the midgut epithelium of susceptible lepidopteran insect species and interferes with midgut function, thus acting as a potent and highly specifi c insecticide (Van Rie et al., 1989). Qualitative detection of genetically modifi ed plants, i.e. diff erentiation of the successfully transformed plants from the non-transformed ones, or modifi ed and non-modifi ed varieties, can be performed on the basis of specifi c po ly me314 T. Vyhnanek, P. Hanacek ra se chain reaction (PCR) for the sites of the used construct (Vaughn et al., 2005). Holck et al. (2002) and Hernandez et al. (2003) reported that they applied PCR methods for the detection of transgenes of maize hybrids derived from MON810. In order to give an accurate quantifi cation of the DNA of the genetically modifi ed plant in a seed sample or in maize-based feed or food products it is necessary to use real-time PCR which is much more demanding in terms of apparatuses and material (Ma et al., 2005; Yang et al., 2005). The objective of the study was to optimise the qualitative and semi-quantitative detection of the 35S CaMV promotor on a model of Bt-maize (MON810) by means of polymerase chain reaction. MATERIAL AND METHODS For qualitative and semi-quantitative detection of genetically modifi ed maize we used two maize genotypes from the company Monsanto, i.e. the MON810-based transgenic hybrid of Bt-maize and genetically non-modifi ed control (isogenic line of Bt-maize). The seeds were the subject matter of a “material transfer agreement” between Monsanto Europe and MUAF in Brno of 26 May 2008. For molecular analysis the genomic DNA was isolated from plants in the stage of one leaf (10-day-old plants, grown at a temperature of 20oC and light regime of 12 h light and 12 h darkness) using the isolation kit DNeasy Plant Mini Kit (Qiagen, GER). The plants were cultivated and genomic DNA isolated in rooms intended for handling GMO. A er isolation the DNA concentration was determined fl uorimetrically. Table I gives the sequences of primers used for the analysis. The reaction mixture of 24 μl contained 30 ng template DNA, 0.5 U Taq polymerase (Promega, USA), 1x corresponding buff er, 7.5 μM of each primer and 0.1 mM of each dNTP. Table II gives the thermal and time profi le for the respective primer combinations. To optimise the semi-quantitative PCR we prepared samples of diff erent concentrations of the genomic DNA of Bt-maize MON810 admixture (1, 5, 10, 25, 50 and 100%) added to the genomic DNA of the isogenic non-transformed line. In the case of semi-quantitative assessment samples of the amplifi cation product were taken between the 18th and 34th PCR cycle. The products were visualised using 1.5% agarose gel electrophoresis (stained with ethidium bromide). The resulting electrophoretograms were digitalised using a CCD camera and the Scion Image programme (USA). RESULTS AND DISCUSSION The fi rst step of implementation of PCR methods for the detection of Bt-maize was to optimise the protocol for detection of the maize genome and of the specifi c site of the genetically modifi ed maize MON810. Many primer combinations have been described and for the detection of the maize genome we selected primers IVR1-F and IVR1-R (invertase gene) (Greiner et al. 2005). A 226 bp product verifi ed the presence of the maize genome (Fig. 1). For quali ta ti ve detection of the insert of Bt-maize MON810 we selected for the detection of the 35S CaMV promotor two primer combinations–BT03/BT04 (Hernandez et al., 2005) and VW01/VW03 (Greiner et al., I: PCR primers used for the amplifi cation of species-specifi c and transgenic DNA of maize Target Primer Amplicon length (bp) Reference Maize (invertase gene) IVR1-F 5 CCG CTG TAT CAC AAG GGC TGG TAC 3 226 Greiner et al. (2005) IVR1-R 5 GGA GCC CGT GTA GAG CAT GAC GAT C3 MON810 (a) (35S-CaMV)* VW01 5 TCG AAG GAC GAA GGA CTC TAA CG3 178 VW03 5 TCC ATC TTT GGG ACC ACT GTC G3 MON810 (b) (35S-CaMV)* BT03 5 TGA TGT GAT ACT TCC ACT GAC G5 280 Hernandez et al. (2005) BT04 5 CGG CAA GTA ATC AGC ACA G3 * 35S-CaMV – 35S promoter from caulifl ower mosaic virus II: PCR programmes used for the detection of species-specifi c and transgenic DNA sequences (Greiner et al., 2005; Hernandez et al., 2005) IVR1-F/IVR1-R VW01/VW03 BT03/BT04 Initial denaturation 10 min, 95 oC 10 min, 95 oC 10 min, 95 oC No. of cycles 40 40 40 Denaturation 30 s, 95 oC 30 s, 95 oC 20 s, 95 oC Annealing 30 s, 64 oC 30 s, 64 oC 30 s, 60 oC Extension 30 s, 72 oC 30 s, 72 oC 60 s, 72 oC Final extension 7 min, 72 oC 7 min, 72 oC 5 min, 72 oC Optimisation of qualitative and semi-quantitative detection of genetically modifi ed crops by PCR 315 2005); the presence of which is verifi ed by a product of the size 280 bp (Fig. 2a) and 178 bp (Fig. 2b). When we used primers and the PCR programme published by Hernandez et al. (2005) we obtained only in one case a weak positive reaction (280 bp product size, Fig. 2a). By contrast, when we used primers and the PCR programme published by Greiner et al. (2005) amplifi cation was successful in all cases (Fig. 2b), and therefore we selected primers VW01 and VW03 for the optimisation of sampling for semi-quantitative PCR. In the fi rst variant of the semi-quantitative analysis we used 18 to 24 cycles of PCR reaction. Product of the size 178 bp was not detected in any used concentration of DNA of Bt-maize in the analysed sample. In the second variant samplings for semi-quantitative analysis were shi ed to the 26th to 34th cycles of the PCR reaction. On the basis of the achieved results we can recommend sampling of the amplifi ca226 bp M 1 2 3 4 5 6 7 M – DNA size marker (pBR322 DNA HaeIII, MBI Fermentas); 1 to 4 – isogenic maize line; 5 to 7 – Bt-maize (MON810) 1: Specific detection of the maize genome with primers IVR–F and IVR 1-R.

Optimisation of qualitative and semi-quantitative detection of genetically modified crops by PCR