Development of a new approach based on real time PCR coupled with high resolution melting (HRM) analysis towards the entomological authentication of honey
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Honey is a natural product widely consumed around the globe, not only for its taste and nutritional value,
but also for its health benefits. Being a product of high dietary relevance and increasing demand, it has
also become a target of economically motivated adulteration. According to the 2014 European Parliament
report on the food crisis, fraud in the food chain and the control thereof, honey is among the 10 food
products most prone of being adulterated [1]. Up until now, honey authenticity was mainly focused on the
issues of sugars addition and botanical and geographical origin. However, recently an increased attention
has been paid to the entomological origin of honey. To this aim, different approaches have been proposed
to differentiate honey produced by different Apis mellifera subspecies, including those from distinct
mitochondrial (mt) DNA lineages [2]. This work aimed to develop a novel real-time PCR method coupled
with HRM analysis that allows for the simultaneous differentiation of honeybee from maternal lineages A,
M and C, for further application in honey authentication. In this sense, data previously obtained from the
mitogenomes of a total of 112 honeybees of different lineages were considered for the development of
new DNA markers. Considering the aim of further application in honey, new primer sets were designed to
amplify short fragments that included different single nucleotide polymorphisms (SNPs) allowing for
HRM application. Three primer sets were proposed, amsCOI-F/amsCOI-R targeting the Cytochrome
oxidase I (COI) gene, amsND1-F-amsND1-R targeting the NADH-ubiquinone oxidoreductase chain 1
(ND1) gene and amsCox3-F/amsCox3-R targeting the Cytochrome oxidase subunit III (Cox3) gene. Each
primer set was first tested using qualitative PCR using DNA extracted from honeybees of A, M and C
mtDNA lineages. After optimizing the real-time PCR conditions, each primer set was tested using a series
of mtDNA extracted from honeybees. While amsCOI-F/amsCOI-R allowed only for the separation of the
honeybees in two clusters, with lineage C and M clustering together, both the amsCox3-F/amsCox3-R and
amsND1-F/amsND1-R set of primers allowed to differentiate the three lineages in separate clusters, with
high level of confidence. As future work, the methodology will be assayed in commercial honey samples.
