Quantification
For quantification of the GM plant material present in a sample, real-time PCR assays are commonly employed to determine the amount of sequence copies of the GM target versus the reference gene target, which obviously is not generating a direct weight-to-weight measurement (ENGL 2007). These assays use standard curves generated with a serial dilution of DNA of known GM content and target sequence concentration. In this way two calibration curves are constructed, one for the targeted GM sequence and one for the plant taxon-specific reference gene. The calibration DNA can be the DNA extracted from certified reference materials or plasmids (Block and Schwarz 2003), or hybrid amplicons carrying both target sequences can be used (Pardigol et al. 2003). The standard curves and the sample DNA are analysed in the same PCR run and, by extrapolating the Ct obtained, quantitative information for the targets is obtained. The copy numbers are calculated for the GM target sequence and the reference gene and used to estimate the relative amount and percentage of the GM plant event present in a given sample. Target DNA copy numbers of standards and quantitative positive controls must be precisely quantified before use, for example by fluorometric techniques (Ahn et al. 1996) or by spectrophotometry analysis (ISO 2005c). The DNA concentration measured is converted to copy number equivalents by using conversion factors, as reported by Arumuganathan and Earle (1991), or by referring to the plant DNA C-value database (Bennett and Leitch 2005). If certified reference materials of a certain percent GM content are used, the percentage of the material must be considered when calculating GM copy number equivalents for these materials. However, it should be also noted that quantitative PCR methods often measure the GM content in relation to specific reference materials, thus the genetic situation (zygosity, degree of ploidy, copy number per genome, etc.) is not considered, which could be an important issue particularly for maize (Papazova et al. 2005a, b; ENGL 2007).
Because of the relatively high measurement uncertainty (MU) accompanied with DNA-based quantitative analysis of the GM plant content in a given sample, it is important that testing laboratories apply procedures to calculate the combined standard deviation accumulating during the whole analytical process. Such a practical approach was recently described for the calculation of the overall MU for decision-making concerning the European 0.9% labelling threshold (Zel et al. 2007). These authors report that, for event GTS-40-3-2 (Roundup Ready soybean, see Chaps. 9, 24), the expanded uncertainty was 23.2%.
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