Browsing by Author "Santos, Joana"
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- 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 authenticate the origin of Mediterranean honeysPublication . Honrado, Mónica; Henriques, Dora; Santos, Joana; Yadró Garcia, Carlos A.; Medibees Consortium; Pinto, M. Alice; Amaral, Joana S.As defined by the Codex Alimentarius, honey is the natural sweet substance produced by honeybees from the nectar of plants.1 This natural product is widely appreciated but is also considered one of the foods most prone to adulteration. The increasing demand for monofloral honey and those with protected designation of origin (PDO) has led to increased fraud by mislabeling botanical and geographical origin.2 Verifying the geographical origin of honey is a challenging endeavor. Recently, attention has been paid to the entomological origin, as it aligns with the geographical patterns of honeybee subspecies. The Mediterranean region is a hot spot of Apis mellifera subspecific diversity shaped by thousands of years of evolution. Although contemporary human-mediated movements of queens have impacted the native subspecific distribution, several PDO honeys specify the subspecies that produce those honeys, thus offering a unique avenue for authentication. As part of the European PRIMA project MEDIBEES, 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, the DNA of 1280 honeybees representing 16 subspecies and the four lineages (A.m. sahariensis, A.m. intermisa, 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. caucasica, A.m. meda, A.m. anatoliaca, A.m. syriaca, A.m. jemenitica, A.m. lamarcki) was extracted, and their whole genomes were sequenced. The MitoZ software was used to assemble the mitochondrial genomes, resulting in 769 mitochondrial genomes successfully assembled. Subsequently, each of these genomes was aligned individually with a reference genome using MEGA software, and mitogenomes not specific to Apis mellifera were discarded. Of these, only the mitogenomes corresponding to the native ancestry were retained, resulting in a final set of 355 mitogenomes in the database. A phylogenetic analysis was conducted with the final 355 mitochondrial sequences, revealing four distinct clusters corresponding to the four maternal lineages. This dataset was used for calculating the fixation index (FST) pairwise values, and a sliding window of 400 bp was used to identify single nucleotide polymorphisms (SNPs) that effectively differentiate (FST>0.98) the four lineages, enabling the identification of promising regions for primer design. This work resulted in the discovery of three promising regions for discriminating the four maternal lineages: one in the COI gene, one in the ND1 gene, and one in the CYTB gene (Fig. 1).
- 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.
- 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.