Quantitative Real-time PCR differentiation between Genetically Modified pollen and Genetically Modified flour in honey

In 2011, the European Court of Justice decided upon the need to demonstrate that pollen is a natural constituent of honey rather than an ingredient. Later, in 2013, the European Parliament defined pollen as a natural constituent of honey. This decision avoided strong financial implications concerning the need of honey labelling whenever Genetically Modified (GM) pollen makes up more than 0.9% of the species pollen fraction, according to the Regulation (EC) 1829/2003. Contrarily, the presence of flour, which is not a natural component of honey but may occur when industrial mills are in the neighbourhood of the hives, has to be treated according to the legal labelling framework for ingredients. Therefore, there is an urgent need to distinguish between pollen and flour. During the last years no helpful technique was found to separate pollen from other kinds of contaminations.
In this work, we investigated the ability of quantitative real-time PCR, associated with plasmid calibrants and Taqman chemistry, to differentiate between an unavoidable presence of GM pollen from a GM flour contamination. A seven point dilution series from a 2x106 plasmid copies/µL solution was used to establish two calibration curves, being one for the transgene and other for a species-specific gene. A good estimate for the relative transgene copy number was obtained for DNA extracted from honey from Madeira Island, a Portuguese GM free zone, spiked either with pollen, flour or embryos from a hemizygous segregating F1 population which had the female progenitor as the transgene donor. Spiked honey was used to minimize the matrix effect. PCR efficiencies were of 94%-99% for both reactions. This approach has considering potential to distinguish pollen from flour and, therefore, to help the national services for the control of labelling and to establish recommendations for the milling companies to minimize the entry of flour into the hives.