A novel real-time PCR coupled with high resolution melting analysis as a simple and fast tool for the entomological authentication of honey by targeting Apis mellifera mitochondrial DNA.

Honey is one of the foods easily adulterated worldwide. Recently, the analysis of honeybee DNA has been proposed as a useful tool to authenticate the entomological origin of honey. However, the methods proposed so far require more than one polymerase chain reaction (PCR) and the use of agarose gels, making the authentication process laborious and lengthy. In this work, a novel real-time PCR coupled with high-resolution melting (HRM) analysis of a 150 bp fragment of the cytochrome c oxidase I (COI) gene is proposed as a fast and simple tool to assess honey's entomological origin by discriminating the mitochondrial DNA lineages of European honey bees (A, M and C lineages). In addition, the new tool allowed the differentiation of honeys produced by different mitotypes of C-lineage ancestry. The method showed high analytical performance and was able to successfully identify the entomological origin of honeys of known origin obtained from research apiaries/beekeepers. Therefore, it was applied to 44 commercial honeys from different countries. It confirmed the entomological authenticity of French PDO honeys that should be produced by the Corse ecotype A. m. mellifera. For the remaining honeys, the results were also in good agreement with the declared geographical origin. However, three honeys from Slovenia did not cluster with C2 mitotype A. m. carnica as expected, suggesting the mixture of honeys produced by honeybees of different mitotypes.