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- DNA-based methods as a powerful tool for the entomological authentication of honeyPublication . Honrado, Mónica; Quaresma, Andreia; Lopes, Ana; Pinto, M. Alice; Henriques, Dora; Amaral, Joana S.Honey is a food widely consumed worldwide and much appreciated for its nutritional, organoleptic and health properties. However, it is also considered one of the food products most prone to be adulterated in the EU. Up until now, honey authenticity addressed mainly the issue of sugars addition and botanical origin. Still, increased attention has recently been paid to honey entomological origin as it also relates to its geographical origin since honeybees carrying mitochondrial DNA (mtDNA) of distinct ancestries can be found across Europe. While in Portugal mtDNA of the autochthonous subspecies Apis mellifera iberiensis belongs to the African (A) lineage, in the northeastern part of Iberia African mitotypes are replaced by mitotypes of western European (M-lineage) ancestry. The native distribution of the M-lineage A. m. mellifera expands from the Pyrenees to Scandinavia and from the British Isles to the Ural Mountains while the C-lineage A. m. ligustica and A. m. carnica subspecies are naturally found in the Apennine and Balkan peninsulas, respectively [1]. Also, certain honeys holding the protected designation of origin (PDO) label should be produced by autochthonous A. mellifera subspecies, as mentioned in their EU geographical indications register.
- COI Metabarcoding as a Novel Approach for Assessing the Honey Bee Source of European HoneyPublication . Honrado, Mónica; Quaresma, Andreia; Henriques, Dora; Pinto, M. Alice; Amaral, Joana S.Honey is a widely consumed food product frequently subjected to adulteration, with the mislabeling of its botanical or geographical origin being one of the most common practices. Determining the entomological origin of honey is particularly challenging but of high relevance for ensuring its authenticity, especially for products with protected designation of origin (PDO) labels. This study presents a novel DNA metabarcoding approach targeting a highly informative 406 bp fragment of the cytochrome c oxidase I (COI) gene to differentiate among the three major mitochondrial lineages (A, M, and C) of honey bees (Apis mellifera L.) native to Europe. The target region was selected based on the calculated fixation index (FST), which is frequently used in Population Genetics as a measure of differentiation between populations. The approach was validated with 11 honey samples of known entomological origin and applied to 44 commercial honeys from 13 countries. The approach demonstrated high sensitivity, accurately identifying the entomological origin of honey, including samples produced by honey bees of varying ancestries, which could not be resolved by previous methods based on real-time PCR coupled with high-resolution melting (PCR-HRM) analysis. The results demonstrate the effectiveness of COI metabarcoding in verifying honey authenticity and highlight the predominance of C-lineage honey bees in the production of commercial honeys from northwestern Europe. This finding suggests a limited presence of the native M-lineage ancestry, underscoring the need for conservation efforts.
- Development of a loop-mediated isothermal amplification assay for the rapid detection of Styphnolobium japonicum (L.) Schott as an adulterant of Ginkgo biloba (L.)Publication . Rodrigues, Vânia; Honrado, Mónica; Santos, Joana; Pinto, M. Alice; Amaral, Joana S.; Rodrigues, VâniaSpecies adulteration is a concern in herbal products, especially when plant substitutes of lower economic value replace valuable botanicals. Styphnolobium japonicum is well known as a potential adulterant of Ginkgo biloba, which is one of the most demanded medicinal plants due to its wide use in pharmaceuticals, food supplements, and traditional medicine. Despite bearing some resemblance to ginkgo's flavonol composition, S. japonicum lacks many of G. biloba's desired therapeutic properties. To prevent adulteration practices, it is crucial to implement rigorous quality control measures, including fast and simple diagnostic tools that can be used on-field. Purpose: This study aims to develop for the first time a species-specific loop-mediated isothermal amplification (LAMP) method for the fast identification of S. japonicum in ginkgo-containing products. Methods: A set of four specific primers (SjF3, SjB3, SjFIP, and SjBIP) and loop primers (SjLF and SjLB) were designed for a LAMP based assay using the 5.8S partial sequence and the internal transcribed spacer 2 of nuclear ribosomal DNA of S. japonicum. Results: The successful amplification of the LAMP assay was inspected through visual detection, with the highest intensity recorded at the optimal conditions set at 68 °C for 40 min. The primers showed high specificity and were able to accurately discriminate S. japonicum from G. biloba and 49 other species of medicinal plants. Furthermore, the proposed LAMP assay proved to be fast, selective, and highly sensitive, as demonstrated by the absolute and relative limits of detection, which were reached at 0.5 pg for S. japonicum DNA and 0.01 % S. japonicum in G. biloba, respectively. Conclusions: This novel approach allows easy identification and discrimination of S. japonicum as a potential adulterant of G. biloba, thus being a useful tool for quality control. Compared to chromatographic or PCR-based methods, the assay proved to be fast, sensitive and did not require expensive equipment, thus offering the possibly usage in field analysis.
- Exploiting the mitogenomes of apis mellifera subspecies to develop an authentication tool to verify the entomological origin of mediterranean honeysPublication . Honrado, Mónica; Henriques, Dora; Santos, Joana; Yadró Garcia, Carlos A.; Martín-Hernández, Raquel; Nanetti, Antonio; González, Amelia Virginia; Al Shagour, Banan; Hosri, Chadi; Farrugia, Dylan; Giovanni, Cilia; Zammit Mangion, Marion; Muz, Mustafa Necati; Haddad, Nizar; Galea, Thomas; Haider, Yamina; Obeidat, Wisam; Aglagane, Abdessamad; Arab, Alireza; Varnava, Andri; Eissa, Asmaa Anwar; Muz, Dilek; Hatjina, Fani; Lamghari, Fouad; Arruda, James; Caristos Caristos, Leonidas; Pinto, M. Alice; Amaral, Joana S.Honey is highly susceptible to adulteration. Currently, the assessment of its geographical origin remains one of the most difficult tasks, which is typically performed by melyssopalynology. Recently, the attention has shifted towards indirect approaches such as the entomological origin based on geographical distribution patterns of honey bee subspecies. Although queens’ trade has impacted the natural subspecies distribution, honeys produced with autochthonous bees or bearing a Protected Designation of Origin specifying the producing honey bee subspecies, offer a unique avenue for authentication. In the MEDIBEES project, we aim to develop a DNA-metabarcoding approach to authenticate honey's entomological origin focusing on mitochondrial lineages A, M, C, and O. To achieve this goal, the DNA from 1251 honey bees representing 16 subspecies (A.m. sahariensis, A.m. intermissa, A.m. siciliana, A.m. ruttneri, A.m. iberiensis, A.m. ligustica, A.m. macedonica, A.m. adami, A.m. cecropia, A.m. cypria, A.m. caucasia, A.m. meda, A.m. anatoliaca, A.m. syriaca, A.m. jemenitica, A.m. lamarcki) was extracted and the whole genome sequenced. From those, 740 mitogenomes were assembled using the MitoZ software. The quality of the assembled mitogenome was assessed by aligning all the sequences using MEGA and 348 samples were deleted. Finally, a phylogenetic analysis was conducted to eliminate non-local subspecies, resulting in a total of 326 mitogenomes. This dataset was used for calculating the fixation index (FST) pairwise values, and a sliding window of 400bp was used to identify single nucleotide polymorphisms that effectively differentiate (FST>0.98) the four lineages, enabling the identification of promising regions for primer design. In this study, three regions were identified that discriminate the four maternal lineages while showing an appropriate length for metabarcoding, namely in the COI, ND1 gene, and CYTB genes.
- Estrutura populacional e estado de conservação das subespécies de Apis mellifera no Oriente Próximo e MédioPublication . Yadró Garcia, Carlos A.; Henriques, Dora; Honrado, Mónica; Amaral, Joana S.; Eissa, Asmaa Anwar; Haddad, Nizar; Obeidat, Wisam; Arruda, James; Lamghari, Fouad; Cilia, Giovanni; Martín-Hernández, Raquel; Nanetti, Antonio; Pinto, M. AliceA abelha melífera, Apis mellifera, é composta por 31 subespécies que se encontram distribuídas na Ásia, África e Europa. O objetivo deste trabalho é desvendar a estrutura populacional e verificar o estado de conservação de três subespécies do Médio Oriente, as quais têm sido pouco estudadas. Para isso, foi extraído o DNA a partir de tóraxes inteiros de machos de 329 amostras de A. m. lamarckii (Egito, 68 amostras), A. m. syriaca (Jordânia, 238 amostras) e A. m. jemenitica (Omã e Emirados Árabes Unidos, 23 amostras). Foram adicionadas 21 amostras de A. m. ligustica, que é uma subespécie amplamente utilizada pelos apicultores no mundo inteiro e por isso fonte de introgressão genética. O genoma completo das 329 amostras foi sequenciado na plataforma Illumina NovaSeq 600 tendo como objetivo uma cobertura de 20X. Os 329 genomas foram mapeados usando o genoma de referência Amel_HAv3.1 e foi implementada uma pipeline que garante a qualidade dos dados. No final, obteve-se um total de 4.030.485 de SNPs que foram usados na reconstrução da estrutura populacional com o ADMIXTURE e PCA. As amostras egípcias mostraram que apesar de terem alguma introgressão de A. m. ligustica, essa não é relevante e é variável (Q-values entre 1E-05 e 0.44), com a maior parte (97%) das amostras apresentando um valor médio de 0.07 ± 0.06 (Q-values, meia ± DP). A. m. syriaca apresenta uma estrutura complexa, tendo sido observados dois grupos distintos pelo PCA e três pelo ADMIXTURE. Relativamente seu ao estado de conservação, foram detetados 76 indivíduos com uma proporção considerável (Q-values entre 0.15 e 0.47) de introgressão com A. m. ligustica. No caso de A. m. jemenitica, foram observados dois cenários diferentes. Em Omã, todas as amostras estudadas mostraram ser puras. Por outro lado, apenas sete amostras dos Emirados Árabes Unidos foram classificadas como tal, enquanto as restantes mostraram proporções de introgressão semelhantes às do Egito. Estes resultados evidenciam o estado precário de integridade genética que estas subespécies apresentam nos locais estudados. No entanto, a existência de indivíduos que podem ser considerados puros para suas respetivas subespécies pode servir como ponto de partida para o desenvolvimento de planos de conservação.
- Semi-automated sequence curation for reliable reference datasets in ITS2 vascular plant DNA (meta-)barcodingPublication . Quaresma, Andreia; Ankenbrand, Markus J.; Garcia, Carlos Ariel Yadró; Rufino, José; Honrado, Mónica; Amaral, Joana S.; Brodschneider, Robert; Brusbardis, Valters; Gratzer, Kristina; Hatjina, Fani; Kilpinen, Ole; Pietropaoli, Marco; Roessink, Ivo; Steen, Jozef van der; Vejsnæs, Flemming; Pinto, M. Alice; Keller, AlexanderOne of the most critical steps for accurate taxonomic identification in DNA (meta)-barcoding is to have an accurate DNA reference sequence dataset for the marker of choice. Therefore, developing such a dataset has been a long-term ambition, especially in the Viridiplantae kingdom. Typically, reference datasets are constructed with sequences downloaded from general public databases, which can carry taxonomic and other relevant errors. Herein, we constructed a curated (i) global dataset, (ii) European crop dataset, and (iii) 27 datasets for the EU countries for the ITS2 barcoding marker of vascular plants. To that end, we first developed a pipeline script that entails (i) an automated curation stage comprising five filters, (ii) manual taxonomic correction for misclassified taxa, and (iii) manual addition of newly sequenced species. The pipeline allows easy updating of the curated datasets. With this approach, 13% of the sequences, corresponding to 7% of species originally imported from GenBank, were discarded. Further, 259 sequences were manually added to the curated global dataset, which now comprises 307,977 sequences of 111,382 plant species.
- Next-generation sequencing as a promising approach for assessing the entomological origin of honeyPublication . Honrado, Mónica; Quaresma, Andreia; Pinto, M. Alice; Henriques, Dora; Amaral, Joana S.Honey is a food widely consumed worldwide and much appreciated for its nutritional and organoleptic properties as well as for its beneficial health effects. However, honey is also considered one of the foods most prone to be adulterated either by the admixing of honey with lower quality, by the addition of sugars, or by mislabeling of botanical and geographical origins, among other possible frauds.1 Therefore, typically, honey authentication has focused mainly on the development of techniques targeting these types of frauds. Recently, increased attention has been paid to honey’s entomological origin since it also relates with geographical origin whose label non-compliances are difficult to detect. Moreover, in the current context where native honeybees are increasingly threatened by introgression, due to the use of exotic queens, preservation of honeybee subspecies in their native ranges, to which they are better adapted, is perceived as of high importance. In this sense, valorisation of the honey produced by native subspecies has been suggested as a possible approach to generate higher income for beekeepers, contributing to the development of rural regions and of sustainable beekeeping based on conservation strategies
- Development of a new approach based on real - time PCR coupled with high resolution melting (HRM) analysis towards the entomological authentication of honeyPublication . Honrado, Mónica; Pinto, M. Alice; Lopes, Ana; Henriques, Dora; Amaral, Joana S.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.
- Animal species authentication in dairy productsPublication . Mafra, Isabel; Honrado, Mónica; Amaral, Joana S.Milk is one of the most important nutritious foods, widely consumed worldwide, either in its natural form or via dairy products. Currently, several economic, health and ethical issues emphasize the need for a more frequent and rigorous quality control of dairy products and the importance of detecting adulterations in these products. For this reason, several conventional and advanced techniques have been proposed, aiming at detecting and quantifying eventual adulterations, preferentially in a rapid, cost-effective, easy to implement, sensitive and specific way. They have relied mostly on electrophoretic, chromatographic and immunoenzymatic techniques. More recently, mass spectrometry, spectroscopic methods (near infrared (NIR), mid infrared (MIR), nuclear magnetic resonance (NMR) and front face fluorescence coupled to chemometrics), DNA analysis (real-time PCR, high-resolution melting analysis, next generation sequencing and droplet digital PCR) and biosensors have been advanced as innovative tools for dairy product authentication. Milk substitution from high-valued species with lower-cost bovine milk is one of the most frequent adulteration practices. Therefore, this review intends to describe the most relevant developments regarding the current and advanced analytical methodologies applied to species authentication of milk and dairy products.
- A sequenciação de nova geração como uma abordagem promissora para a identificação da origem entomológica do melPublication . Honrado, Mónica; Henriques, Dora; Yadró Garcia, Carlos A.; Santos, Joana; Rufino, José; Medibees Consortium; Pinto, M. Alice; Amaral, Joana S.O mel é um alimento muito consumido e apreciado em todo o mundo pelas suas propriedades nutricionais e organoléticas, bem como pelos seus efeitos benéficos para a saúde. No entanto, é também considerado um dos alimentos mais suscetíveis de ser adulterado, quer pela mistura de mel de qualidade inferior, quer pela adição de açúcares, ou pela rotulagem incorreta da origem botânica e/ou geográfica, entre outras possíveis fraudes. Nos últimos anos, tem sido dada uma atenção crescente à origem entomológica do mel, uma vez que esta também está relacionada com a origem geográfica. No âmbito do projeto PRIMA “MEDIBEES” (https://medibees.org/), a sequenciação de nova geração (NGS) será utilizada com vista ao desenvolvimento de ferramentas moleculares que permitam identificar a origem entomológica de amostras de mel provenientes dos 8 países mediterrânicos do consórcio, de forma a diferenciar e valorizar méis produzidos por abelhas autóctones destes países. Com este objetivo, inicialmente procedeu-se à construção da base de dados das sequências de DNA mitocondrial das abelhas de modo a incluir 10 subespécies mediterrânicas das 4 linhagens maternas (A, M, C e O). Para tal, procedeu-se à extração de DNA e à respetiva sequenciação dos genomas completos, na plataforma Illumina Novaseq 6000, de um total de 1095 abelhas destes países. Posteriormente, utilizou-se o programa mitoZ 3.6 para fazer a montagem do genoma mitocondrial de cada uma das amostras, resultando na seleção de 283 sequências mitocondriais com boa montagem. Em seguida, foi utilizado o software MEGA 11, para realizar o alinhamento destas sequências. A informação obtida será posteriormente utilizada para a seleção de regiões com variantes (SNPs) informativos que possam ser usadas para o desenho de primers adequados e desenvolvimento de ferramentas para a identificação de méis produzidos por abelhas de diferentes linhagens mitocondriais e respetivas subespécies.