In-house Validation of a DNA Extraction Protocol from Honey and Bee Pollen and Analysis in Fast Real-Time PCR of Commercial Honey Samples Using a Knowledge-Based Approach
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For consumers, honey is a natural product that should not be subjected to treatment or alteration. Since the question of the presence of genetically modified organisms concerned pollen in honey, the aim of this research was to find an alternative, however, practical and efficient method of honey and bee pollen DNA extraction for routine analysis application. Furthermore, to evaluate the extracted DNA, a real-time PCR system based on the actin gene was optimized and validated in fast mode for the first time to reduce analysis time. To develop an alternative DNA extraction protocol, two already published procedures were combined and tested with some variations, in particular without beads/filters used to grind/enrich pollen. The best approach found in terms of quantity and quality of extracted DNA was a combination of the pretreatment and the extraction method, described in the German guideline, with some modifications and the addition of a DNA purification kit. This protocol was validated with DNA extracts from honey and bee pollen and it was applied to 18 commercial honey samples. Furthermore, a sample proved positive in transgenic screening elements analysis and for transgenic event identification, a knowledge-based approach was adopted. Since the DNA extraction protocol proved suitable, it could be applied for other analysis such as molecular species characterization, the study of traceability, and environmental monitoring, considering honey as a vector of authorized and not authorized genetically modified organisms.
KeywordsHoney Pollen DNA extraction actin gene Fast real-time PCR Genetically modified organism (GMO) Knowledge-based approach
The study was financed by the Italian Ministry of Health grant IZSUM 14/2012 RC (“Sviluppo di metodiche biomolecolari per la rilevazione di OGM nel miele” “Development of biomolecular methods for GMO identification in honey”).
The authors would like to thank “Anabolic and Residues Laboratory” and “Environmental Contaminants Laboratory” of Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche that provided commercial honey/honeycomb samples.
Compliance with Ethical Standards
Conflict of Interest
Martina Torricelli has no conflict of interest. Elisa Pierboni has no conflict of interest. Gloria Raquel Tovo has no conflict of interest. Ludovica Curcio has no conflict of interest. Cristina Rondini has no conflict of interest.
This article does not contain any studies with human participants or animals performed by any of the authors.
Informed consent is not applicable.
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