DNA-Based Method for Traceability and Authentication of Apis cerana and A. dorsata Honey (Hymenoptera: Apidae), Using the NADH dehydrogenase 2 Gene

Abstract

Honey is a widely used natural product and the price of honey from Apis cerana (ACH) and A. dorsata (ADH) is several times more expensive than the one from A. mellifera (AMH), thus there are increasing fraud issues reported in the market by mislabeling or mixing honeys with different entomological origins. In this study, three species-specific primers, targeting the NADH dehydrogenase 2 (ND2) region of honeybee mitochondrial DNA, were designed and tested to distinguish the entomological origin of ACH, ADH, and AMH. Molecular analysis showed that each primer set can specifically detect the ND2 region from the targeted honeybee DNA, but not from the others. The amplicon size for A. cerana, A. dorsata and A. mellifera were 224, 302, and 377 bp, respectively. Importantly, each primer set also specifically produced amplicons with expected size from the DNA prepared from honey samples with different entomological origins. The PCR adulteration test allowed detection of 1% of AMH in the mixture with either ACH or ADH. Furthermore, real-time PCR and melting curve analysis indicated the possible discrimination of origin of honey samples. Therefore, we provide the newly developed PCR-based method that can be used to determine the entomological origin of the three kinds of honey.

Honey Quality Control: Review of Methodologies for Determining Entomological Origin

“…Later, Soares et al [ 11 ] designed species-specific primers targeting 111 bp of the tRNAleu-cox2 intergenic region to detect A. cerana DNA through PCR. Recently, Mohamadzade Namin et al [ 63 ] developed three sets of species-specific primers targeting NADH Dehydrogenase 2 (ND2) region of mtDNA ( Table 2 ) in order to distinguish entomological origin of major types of honey in Asian market from A. cerana , A. dorsata , and A. mellifera . The results of the specificity test indicated the possibility of differentiation of honey samples based on gel electrophoresis pattern (223, 301, and 376 bp for A. cerana , A. dorsata , and A. mellifera , respectively).…”

“…The species-specific primers developed by Zhang et al [ 10 ] to distinguish A. cerana honey and A. mellifera honey through gel electrophoresis pattern are also applicable in Real-Time PCR assay through melting curve analysis as a single melt peak for each species was observed (76.4 °C for A. cerana and 82.9 °C for A. mellifera ). In addition, the melting curve analysis demonstrated the possibility of using the primer sets developed by Mohamadzade Namin et al [ 63 ] to identify the entomological origin of honey from A. cerana (Tm: 71.9 °C), A. dorsata (Tm: 69.2 °C), and A. mellifera (Tm: 72.4 °C). Another set of primer was developed by Soares et al [ 11 ] to amplify 140 bp of 16S rRNA gene of the mtDNA to differentiate A. cerana and A. mellifera honey through high-resolution melting analysis of Real-time PCR.…”

Authenticity of Honey: Characterization, Bioactivities and Sensorial Properties

“…Finally, this Special Issue includes a study on the importance of differentiating the entomological origin of honey [12]. Apis mellifera honey, A. cerana honey and A. dorsata honey are the three dominant honey types in the Asian market.…”

“…Some studies indicate that A. dorsata honey has higher antioxidant properties and medical values than A. mellifera honey. A. dorsata honey and A. cerana honey are vulnerable to adulteration (as a result of false geographical and botanical origin, or from mixing their composition with sugars or syrups), due to the higher price in the market compared to A. mellifera honey [12]. Therefore, it is important to develop fast, reliable and cost-effective identification methods to determine the honey's origin.…”

“…Therefore, it is important to develop fast, reliable and cost-effective identification methods to determine the honey's origin. Mohamadzade et al [12] proposed a rapid and accurate PCR-based method. In this study, three species-specific primers were designed to amplify the short part of the NADH dehydrogenase 2 (ND2) region of mtDNA.…”