Analytical MethodsAutomated DNA extraction from pollen in honey
Introduction
Bees process nectar with enzymes before converting it to honey by reducing the water content. Honey is used for human consumption, has medical benefits and is used for cosmetic purposes. However, honey is also a potential risk for human health due to environmental contaminations like pesticides, antibiotics, microbes or heavy metals (Corsini et al., 2013, Martel et al., 2007, Nevas et al., 2002, Ortelli et al., 2004). Ingestion of pollen, as a characteristic component of honey, may also lead to allergic reactions (Helbling, Peter, Berchtold, Bogdanov, & Müller, 1992).
Therefore, honey is subject to morphological, chemical and molecular biological investigations. Analysis of floral origin by microscopic analysis of pollen (Louveaux et al., 1978, Senyuva et al., 2009) is still state of the art. Laube et al. developed a DNA-based method for characterisation of the geographical origin of honey (Laube et al., 2012) and DNA-based methods may also be used to detect allergens and microbes in honey (Olivieri, Marota, Rollo, & Luciani, 2012).
Pollen from genetically engineered (GE) plants may also be present in honey. Until 2011, this had no effect on marketing issues or labelling of honey and honey products. However in September 2011, the Court of Justice of the EU declared pollen to be an ingredient and not a characteristic component of honey as before. As a consequence, honey that contains pollen of a GE plant, which is not authorised as food ingredient, must be removed from the EU market. In case of an authorised GE plant, the honey must be labelled above a GE content of 0.9% (Regulation (EC) No 1829/2003 on genetically modified food and feed, 2003a; Regulation (EC) No 1830/2003 concerning the traceability and labelling of genetically modified organisms and the traceability of food and feed products produced from genetically modified organisms and amending 50 Directive 2001/18/EC, 2003b). Currently, surveillance of honey and honey products for GE contamination is performed by DNA analysis.
DNA analysis requires an efficient DNA extraction method in order to detect minute amounts of DNA, e.g. DNA from allergens or GE plants. Waiblinger et al. (2005) developed a manual CTAB buffer-based DNA extraction procedure, which was further optimised in 2012 (Waiblinger et al., 2012). However, this method is very laborious, contains the use of chloroform and requires a subsequent purification step by using a commercial kit. In order to resolve these negative aspects of this manual DNA extraction procedure, we modified a previously developed automated DNA extraction method (Guertler et al., 2013) to cope with honey as a very complex matrix. An extraction method for DNA from honey must feature a rapid and easy workflow and should result in DNA of high yield and quality. It is also essential to remove inhibitors in order to accomplish a reliable and reproducible PCR analysis.
Section snippets
Sample material
For assay validation, eight different honey samples (one pine honey, two wild honey, five polyflora honey) were pooled in order to gain enough sample material for DNA extraction and method validation. Honey samples were provided by the Bavarian Health and Food Safety Authority (Oberschleißheim, Germany). Further, twelve routine honey samples were analysed in order to check the applicability of the automated DNA extraction in the official honey surveillance.
Prior DNA extraction, honey was
Workflow of the automated DNA extraction
The optimisation process led to the following automated DNA extraction protocol: four 50 ml Falcon tubes are filled with 12.5 g honey. Additionally, 45 ml H2O are added to each tube. The honey is dissolved by shaking the tubes, followed by a centrifugation step for 15 min at 4.000 × g. The supernatant is discarded and the pellet is solved in 5 ml H2O. Subsequently, all 4 tubes for each sample are pooled and filled up with H2O to a total volume of 30 ml. After centrifugation for 10 min at 4.000 × g, the
Discussion
More and more laboratories are engaged in DNA analysis of honey samples with regard to GMO detection, confirmation of origin of honey, detection of microorganisms or determination of allergic components. The increasing need for rapid and standardised DNA extraction protocols brought us to the development of an automated DNA extraction. Currently, DNA is mostly isolated by using a CTAB-buffer based DNA extraction, which is laborious and time-consuming. By altering extraction conditions and
Acknowledgements
Promega GmbH is particularly acknowledged for providing Maxwell® 16 kits.
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