Elsevier

Food Chemistry

Volume 149, 15 April 2014, Pages 302-306
Food Chemistry

Analytical Methods
Automated DNA extraction from pollen in honey

https://doi.org/10.1016/j.foodchem.2013.10.129Get rights and content

Highlights

  • We developed an automated DNA extraction from pollen in honey.

  • We compared this automated DNA extraction procedure with a common manuel DNA extraction method.

  • Automated DNA extraction is fast, easy to handle, reproducible and results in higher DNA yields.

  • This method can be used for any application dealing with DNA analysis in honey.

Abstract

In recent years, honey has become subject of DNA analysis due to potential risks evoked by microorganisms, allergens or genetically modified organisms. However, so far, only a few DNA extraction procedures are available, mostly time-consuming and laborious. Therefore, we developed an automated DNA extraction method from pollen in honey based on a CTAB buffer-based DNA extraction using the Maxwell 16 instrument and the Maxwell 16 FFS Nucleic Acid Extraction System, Custom-Kit. We altered several components and extraction parameters and compared the optimised method with a manual CTAB buffer-based DNA isolation method. The automated DNA extraction was faster and resulted in higher DNA yield and sufficient DNA purity. Real-time PCR results obtained after automated DNA extraction are comparable to results after manual DNA extraction. No PCR inhibition was observed. The applicability of this method was further successfully confirmed by analysis of different routine honey samples.

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.

References (16)

  • E. Corsini et al.

    Pesticide induced immunotoxicity in humans: a comprehensive review of the existing evidence

    Toxicology

    (2013)
  • M. Nevas et al.

    High prevalence of Clostridium botulinum types A and B in honey samples detected by polymerase chain reaction

    International Journal of Food Microbiology

    (2002)
  • S.A. Bustin et al.

    The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments

    Clinical Chemistry

    (2009)
  • P. Guertler et al.

    Development of a CTAB buffer-based automated gDNA extraction method for the surveillance of GMO in seed

    European Food Research and Technology

    (2013)
  • A. Helbling et al.

    Allergy to honey: relation to pollen and honey bee allergy

    Allergy

    (1992)
  • I. Laube et al.

    Development of primer and probe sets for the detection of plant species in honey

    Food Chemistry

    (2012)
  • H. Liu et al.

    Performance evaluation of the Maxwell 16 System for extraction of influenza virus RNA from diverse samples. [Research Support, Non-U.S. Gov’t]

    PLoS One

    (2012)
  • J. Louveaux et al.

    Methods of melissopalynology

    Bee World

    (1978)
There are more references available in the full text version of this article.

Cited by (17)

  • Detection of honey adulteration by conventional and real-time PCR

    2019, Food Control
    Citation Excerpt :

    These methodologies have been successfully applied for the authentication of animal products like milk (Mayer, 2005), meat (Cai, Gu, Scalan, Ramatlapeng & Lively, 2012; Chen, Wei, Chen, Zhao, & Yang, 2015; Farrokhi & Jafari Joozani, 2011; Kesmen, Yetiman, Sahin, & Yetim, 2012; Rodríguez-Ramírez, González-Córdova, & Vallejo-Cordoba, 2011; Safdar, Junejo, Arman & Abasiyanik, 2014) and seafood (Fernandes, Costa, Oliveira, & Mafra, 2017; Nebola, Borilova, & Kasalova, 2010; Rasmussen, Morrissey, & Walsh, 2010; Rodríguez-Ramírez et al., 2011). Specifically, in honey, this technique has only been used for the botanical origin identification (Guertle, Eicheldinger, Muschler, Goerlich & Busch, 2014; Laube et al., 2010; Soares, Amaral, Oliveira, & Mafra, 2015). Regarding the positive results obtained in the detection of adulterations in products of animal origin, it could be considered viable that this technique can be applied to other animal by-products like honey.

  • Improving DNA isolation from honey for the botanical origin identification

    2015, Food Control
    Citation Excerpt :

    The use of DNeasy Blood and Tissue Kit (Qiagen GmbH) was reported in other works aiming at detecting DNA from different plant species in honey samples (Laube et al., 2010; Valentini, Miquel, & Taberlet, 2010). Recently, Guertler, Eicheldinger, Muschler, Goerlich, and Busch (2014) reported the development of an automated DNA extraction method from pollen in honey and compared its performance with a manual CTAB buffer-based DNA isolation method. Although the automated method proved to be faster than the manual and resulted in higher DNA yield, it requires the use of high-cost instrumentation.

View all citing articles on Scopus
View full text