Distortion of Genetically Modified Organism Quantification in Processed Foods:  Influence of Particle Size Compositions and Heat-Induced DNA Degradation

Abstract

Milling fractions from conventional and transgenic corn were prepared at laboratory scale and used to study the influence of sample composition and heat-induced DNA degradation on the relative quantification of genetically modified organisms (GMO) in food products. Particle size distributions of the obtained fractions (coarse grits, regular grits, meal, and flour) were characterized using a laser diffraction system. The application of two DNA isolation protocols revealed a strong correlation between the degree of comminution of the milling fractions and the DNA yield in the extracts. Mixtures of milling fractions from conventional and transgenic material (1%) were prepared and analyzed via real-time polymerase chain reaction. Accurate quantification of the adjusted GMO content was only possible in mixtures containing conventional and transgenic material in the form of analogous milling fractions, whereas mixtures of fractions exhibiting different particle size distributions delivered significantly over- and underestimated GMO contents depending on their compositions. The process of heat-induced nucleic acid degradation was followed by applying two established quantitative assays showing differences between the lengths of the recombinant and reference target sequences (A, deltal(A) = -25 bp; B, deltal(B) = +16 bp; values related to the amplicon length of the reference gene). Data obtained by the application of method A resulted in underestimated recoveries of GMO contents in the samples of heat-treated products, reflecting the favored degradation of the longer target sequence used for the detection of the transgene. In contrast, data yielded by the application of method B resulted in increasingly overestimated recoveries of GMO contents. The results show how commonly used food technological processes may lead to distortions in the results of quantitative GMO analyses.

Effect of high temperature and pressure on quantification of MON 810 maize

“…PCR analysis of the sterilised (100 and 121°C) samples revealed a reduction of the extracted DNA size in a time dependent manner and different conditions such as pH and increased pressure (Kollárovič et al 2005;Moreano et al 2005;Hrnčírová et al 2008). A similar effect of baking on the DNA integrity was also described previously (Kollárovič et al 2005;Moreano et al 2005;Gryson et al 2008;Hrnčírová et al 2008).…”

Combined effects of temperature, pressure and low pH on the amplification of DNA of plant derived foods

“…30 min 2004; Moreano et al 2005;Gryson et al 2008;ISO 2005) of GM and non-GM plant samples. PCR analysis of the sterilised (100°C) and autoclaved (120°C) samples revealed the reduction of the extracted DNA size in the time dependent manner under different pH and pressure conditions which is in good agreement with the previous findings (Kollárovič et al 2005;Moreano et al 2005;Hrnčírová et al 2008). PCR primers for different sizes of amplicons were applied for the monitoring of DNA degradation.…”

Suitable method for simultaneous and specific detection of maize (<i>Zea mays</i> l.) and genetically modified soyabean (<i>Glycine max</i> l.) in animal feeds

“…The real time PCR technique is widely known as the most sensitive and highly specifi c method for GMO quantifi cation. Nevertheless, processed feed suffer treatments like grinding and heated up at high temperatures which can distortion the quantifi cation of GMO in the samples (Moreano et al, 2005). In this sense, we have compared both methods, conventional and proposed multiplex RT-PCR, and comparable results were obtained without signifi cant differences for raw and processed feed.…”