Evaluation of the reliability of maize reference assays for GMO quantification.

A reliable PCR reference assay for relative genetically modified organism (GMO) quantification must be specific for the target taxon and amplify uniformly along the commercialised varieties within the considered taxon. Different reference assays for maize (Zea mays L.) are used in official methods for GMO quantification. In this study, we evaluated the reliability of eight existing maize reference assays, four of which are used in combination with an event-specific polymerase chain reaction (PCR) assay validated and published by the Community Reference Laboratory (CRL). We analysed the nucleotide sequence variation in the target genomic regions in a broad range of transgenic and conventional varieties and lines: MON 810 varieties cultivated in Spain and conventional varieties from various geographical origins and breeding history. In addition, the reliability of the assays was evaluated based on their PCR amplification performance. A single base pair substitution, corresponding to a single nucleotide polymorphism (SNP) reported in an earlier study, was observed in the forward primer of one of the studied alcohol dehydrogenase 1 (Adh1) (70) assays in a large number of varieties. The SNP presence is consistent with a poor PCR performance observed for this assay along the tested varieties. The obtained data show that the Adh1 (70) assay used in the official CRL NK603 assay is unreliable. Based on our results from both the nucleotide stability study and the PCR performance test, we can conclude that the Adh1 (136) reference assay (T25 and Bt11 assays) as well as the tested high mobility group protein gene assay, which also form parts of CRL methods for quantification, are highly reliable. Despite the observed uniformity in the nucleotide sequence of the invertase gene assay, the PCR performance test reveals that this target sequence might occur in more than one copy. Finally, although currently not forming a part of official quantification methods, zein and SSIIb assays are found to be highly reliable in terms of nucleotide stability and PCR performance and are proposed as good alternative targets for a reference assay for maize.

Impact of genetic structures on haploid genome-based quantification of genetically modified DNA: theoretical considerations, experimental data in MON 810 maize kernels (Zea mays L.) and some practical applications.

Real-time Polymerase Chain Reaction (PCR) based assays are widely used to estimate the content of genetically modified (GM) materials in food, feed and seed. It has been known that the genetic structures of the analyte can significantly influence the GM content expressed by the haploid genome (HG) % estimated using real-time PCR assays; this kind of influence is also understood as the impact of biological factors. The influence was first simulated at theoretical level using maize as a model. We then experimentally assessed the impact of biological factors on quantitative results, analysing by quantitative real-time PCR six maize MON 810 hybrid kernels with different genetic structures: (1) hemizygous from transgenic male parent, (2) hemizygous from transgenic female parent and (3) homozygous at the transgenic locus. The results obtained in the present study showed clear influences of biological factors on GM DNA quantification: 1% of GM materials by weight (wt) for the three genetic structures contained 0.39, 0.55 and 1.0% of GM DNA by HG respectively, from quantitative real-time PCR analyses. The relationships between GM wt% and GM HG% can be empirically established as: (1) in the case of the presence of a single GM trait: GM HG% = GM wt% x (0.5 +/- 0.167Y), where Y is the endosperm DNA content (%) in the total DNA of a maize kernel, (2) in the case of the presence of multiple GM traits: GM HG% = N x GM wt% x (0.5 +/- 0.167Y), where N is the number of GM traits (stacked or not) present in an unknown sample. This finding can be used by stakeholders related to GMO for empirical prediction from one unit of expression to another in the monitoring of seed and grain production chains. Practical equations have also been suggested for haploid copy number calculations, using hemizygous GM materials for calibration curves.