Tracking adulteration of nectar honey varieties using a high-resolution melting qPCR technique validated with melissopalinology

Honey is a highly-valued food product, appreciated due to its organoleptic and health-promoting properties; which both depend on its botanical provenance. In this study, a collection of nectar honey varieties (acacia, buckwheat, linden, rape, and heather) was subjected to extensive physicochemical, melissopalynological, and molecular analyses, to verify if the latter (qPCR-HRM) can be used as a rapid method for determination of the product's authenticity. To this end, the DNA extracted from the honey pollen grains was subjected to a carefully pre-optimized qPCR-HRM analysis. The effectiveness of extracting DNA templates relied on various factors but a significant role played honey “pollination” degree. The plastidial rbcL barcode was found to be a better option for the taxonomic differentiation with qPCR-HRM when compared to the nuclear targets. The results showed that qPCR-HRM correctly clustered honey samples of the same botanical origin determined by melissopalynology. The qPCR-HRM clustering results were additionally verified by rbcL region sequencing. The most consistent data were obtained for heather honey with over-represented pollen, which results from the generally observed low availability of plastidial DNA in the total DNA extracts. The qPCR-HRM revealed a biological divergence within the honey samples marketed as “acacia”, as confirmed by melissopalynology.