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This paper focuses on the determination of the GMO content of maize and soybean samples using real-time PCR, comparing simplex and duplex PCR. The total DNA content of samples was determined by amplifying part of a maize gene encoding a lipid transfer protein, or part of a soybean lectin gene. The transgenic DNA was quantified by amplifying part of the CaMV 35S promoter. The importance of preparation and conservation of standards as well as the relevance of DNA extraction protocol on the variability of results are discussed. For the determination of low GMO content, limitation in the number of copies of the target gene to be amplified is considered. For samples with a theoretical GMO content of 1%, corresponding to the legal threshold for labelling, the value determined by duplex real-time PCR ranged from 0.85% to 1.20%. Both real-time simplex and duplex PCRs allowed identification of GMO free samples without ambiguity.
Food Control 06/2003; 13(4-5-13):235-244. DOI:10.1016/S0956-7135(02)00015-4 · 2.82 Impact Factor
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Cereal Chem. 79(4):553-558 Common wheat adulteration of durum wheat pasta was quantified using real-time duplex polymerase chain reaction (PCR). The total DNA content of pasta was determined by amplifying part of a wheat gene en- coding a lipid transfer protein, and common wheat DNA was quantified by amplifying part of the puroindoline-b gene. Under the conditions defined by this study, for pasta with a theoretical adulteration of 3%, the experi- mentally determined mean value was 2.6-3.4%, depending on drying tem- perature. Pure durum wheat pastas were distinguished from adulterated pastas without ambiguity. This study demonstrates the feasibility of using real- time duplex PCR to quantify common wheat adulteration of pasta dried at high temperature, quantification that was impossible with the French official peroxidase-marker method. French legislation stipulates (1934) that pasta must be made exclusively from Triticum durum semolina and water, although a 1955 regulation authorizes the addition of salt, eggs, gluten, milk, vegetables or vegetable extracts, and spices. Addition of T. aestivum (common wheat) is the most common adulteration in industrially made pastas. In Spain and Italy, pasta must also have been made exclusively with durum wheat, whereas in north European countries, pastas made with common and durum wheat are permitted. In France, labeling of such mixed products must clearly indicate their com- position and the label "alimentary pasta" may not be used, as this description is reserved for pure durum wheat pasta. However, because of the possibility of accidental contamination occurring during either wheat harvest or storage and transport of grains and semolina, pasta is only officially regarded as impure when the common wheat level exceeds 3%. This legal threshold of 3% is also applicable for export of durum wheat pasta outside the European community as stipulated by the European Commission regulation (1222/94, EC 1994). Different techniques have been used to determine the level of common wheat adulteration in pasta. These have been based on research concerning sitosterol palmitate (Matveff 1952) or water- soluble proteins specific to common wheat (Resmini 1968; Garcia- Faure et al 1969; Feillet and Kobrehel 1972). Unfortunately, these methods are either not specific enough or not sensitive enough. A great improvement was achieved with the method of Kobrehel and Feillet (1976), based on the detection of the peroxidase-a7D specific to the D genome (Kobrehel and Gautier 1974). However, over the last 15 years, a significant modification in pasta technology has occurred: use of high temperatures in the drying process. Indeed, most industrially made pastas are now dried at high or very high temperatures (70-100°C). This results in protein degradation that makes quantification of the level of adulteration impossible with previous methods. In response to this development, new methods based on the identification of T. aestivum specific gliadins (Kobrehel et al 1985; McCarthy et al 1990) or immunodetection of friabilin (Durotest, Rhone Poulenc Diagnostic Ltd.) have been proposed, but these do not meet the standards required for adoption as official methods. Consequently, the Kobrehel and Feillet method, based on the detection of a peroxidase specific to the D genome, is still the official method in France (Journal Officiel 1975). Recently, because DNA exhibits greater thermal resistance than proteins, a polymerase chain reaction (PCR) based method has been developed to detect the simple presence of common wheat in durum wheat