COMBINING MULTIPLE DNA-BASED ASSAYS TO UNCOVER ADULTERATED HONEY

One of the major challenges facing the global beekeeping sector is the widespread presence of honey that does not meet regulatory standards, as evidenced by numerous cases of adulteration. The most common type of fraud involves the addition of sugar syrups to dilute honey. Other fraudulent practices include mislabeling the geographical, botanical, or entomological origin of honey. Because honey fraud is complex and requires multiple types of information to be properly identified, a multi-analytical strategy is necessary. In this study, we developed and employed a combination of DNA-based analytical methods to examine authentic, suspicious, and adulterated honey samples from different countries. The applied methodologies included: i) two targeted DNA-based assays to detect the honey bee mitochondrial DNA lineages that produced the investigated honey; ii) targeted DNA-based assays to detect the varieties/lines of the botanical species from which the honey was prevalently produced; iii) DNA metabarcoding methods to disclose the botanical profiles of honey using seven plant derived universal primer pairs targeting six different gene regions (matK, psbA-trnH, rbcl, trnL_UAA, ITS2 and 18S); iv) DNA metabarcoding methods to define the plant-sucking insect fingerprints of honey using two mitochondrial DNA gene targets (COX1 and CYTB); v) definition of honey microbiota profiles using two 16S primer pairs; vi) three whole DNA sequencing approaches based on different next-generation sequencing techniques. The combined results of these complementary approaches enabled the precise classification of honey samples as either authentic or adulterated. These findings underscore the significance of employing multi-level DNA-based analytical approaches to capture relevant DNA fingerprints of honey which are useful to highlight various types of honey adulterations. Ongoing activities are directed towards the construction of comprehensive reference sequence databases useful to enhance the identification of counterfeit honey by applying various DNA-based assays.