Percorrer por autor "Duc, Hanh Pham"
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- Development of molecular markers for honey entomological origin identificationPublication . Soares, Sónia; Grazina, Liliana; Mafra, Isabel; Costa, Joana; Pinto, M. Alice; Duc, Hanh Pham; Oliveira, Beatriz; Amaral, Joana S.Honey is the natural sweet substance produced by honey bees. According to the European Union legislation, it should be produced by the westem honey bee, Apis mellifera. However, in Ásia, honey is traditionally obtained from other bee species, mainly the eastera honey bee Apis cerana. So far, only a few protein-based methods have been proposed to assess honey entomological origin[ ], which in fact is related to its geographical origin since bee species generally occupy different geographical ranges according to their evolutionary lineages [ ]. In this work, DNA markers were developed for the specifíc identification ofA. mellifera and A. cerana in honey. For this purpose, bees of A. cerana from Thailand, China and Vietnam and honey bees of 4 different subspecies of A. mellifera (iberiensis, mellifera, ligustíca, carnica) from EU countries were used. Different sets ofprimers were designed targeting the 16S rRNA gene and the tRNAleu - COII intergenic region. The specificity and sensitivity of the designed primers were assayed by qualitative polymerase chain reaction (PCR). Primers targeting the intergenic region successfully differentiated A. cerana from A. mellifera. Positive amplifications were obtained for ali the bees with 16S rRNA primers. However, the use of real-time PCR coupled with High Resolution Melting analysis allowed the separation of the two honey bee species in different clusters. The developed methodologies were applied to the analysis ofauthentic honey samples from Vietnam (produced from A. cerana and A. mellifera bees) and from Portugal allowing its successful entomological origin identification.
- Entomological authentication of honey based on DNA markers: differentiation of honey produced by Apis mellifera and Apis ceranaPublication . Amaral, Joana S.; Soares, Sónia; Grazina, Liliana; Mafra, Isabel; Costa, Joana; Pinto, M. Alice; Duc, Hanh Pham; Oliveira, BeatrizAccording to the European Union legislation, honey is the natural sweet substance produced by Apis mellifera, also known as European honeybee. However, in other regions of the world, honey is traditionally obtained from other bee species. Among those, A. cerana (also known as Asian honeybee) is also of economic importance since it is used in apiculture. Due to the decline of the wild populations of the A. cerana in some countries, such as Japan and parts of China, there is an increasingly interest in preserving the native Asian honeybee, being its honey increasingly valued. Owing to the growing demand for this traditional product, the honey produced by A. cerana attains a much higher market value compared to that of A. mellifera, thus being prone to adulteration. So far, only a few protein-based methods have been proposed to assess honey entomological origin [1], which in fact is related to its geographical origin since bee species generally occupy different geographical ranges according to their evolutionary lineages [2]. In this work, DNA methods were developed for the specific identification of A. mellifera and A. cerana DNA in honey. For this purpose, bees of A. cerana from Thailand, China and Vietnam and honeybees of 4 different subspecies of A. mellifera (iberiensis, mellifera, ligustica and carnica) from EU countries were used. Different sets of primers were designed targeting the tRNAleu - COII intergenic region and the 16S rRNA gene. For both cases, the specificity and sensitivity of the designed primers were assayed by qualitative polymerase chain reaction (PCR). DNA was extracted from honey samples as previously described [3]. PCR with primers targeting the tRNAleu - COII intergenic region allowed the specific detection of A. cerana. The applicability of the proposed new PCR method was assayed with authentic A. cerana and A. mellifera honey samples, which enabled the identification of A. cerana honey. PCR targeting the 16S rRNA gene successfully amplified both honeybee species, but without being able to differentiate them. However, the use of real-time PCR with 16S rRNA primers coupled with High Resolution Melting (HRM) analysis allowed the differentiation of both species in distinct clusters (Fig. 1). The developed new HRM methodology was further applied to the analysis of authentic honey samples from Vietnam (produced from A. cerana and A. mellifera honeybees) and from Portugal (produced from A. mellifera honeybees), as well as commercial samples of honey labelled as produced in the EU, allowing its successful entomological origin identification [4]. Both developed techniques proved their effectiveness for establishing the entomological origin of honey and can be considered as useful tools for authentication/control purposes.
- Novel diagnostic tools for Asian (Apis cerana) and European (Apis mellifera) honey authenticationPublication . Soares, Sónia; Grazina, Liliana; Mafra, Isabel; Costa, Joana; Pinto, M. Alice; Duc, Hanh Pham; Oliveira, Beatriz; Amaral, Joana S.Honey can be produced by different species of honeybees, with two being of economic importance due to their use in apiculture, namely Apis mellifera (known as European honeybee) and Apis cerana (known as Asian honeybee). Due to the decline of the wild populations of the Asian honeybee, this honey generally attains much higher market value, being prone to adulteration. This work aims at proposing new tools, based on the use of molecular markers, for the entomological authentication of honey. To this end, new species-specific primers were designed targeting the tRNA leu -cox2 intergenic region and allowing the detection of A. cerana DNA by qualitative polymerase chain reaction (PCR). Additionally, a novel real-time PCR method with high resolution melting analysis was developed to target the 16S rRNA gene of both bee species, allowing their discrimination in different clusters. The proposed methodologies were further applied with success in the authentication of Asian and European honey samples by the identification of honeybee DNA, demonstrating the usefulness of these simple and cost-effective new approaches.