Development of a Real-Time PCR method for the identification of Atlantic mackerel (Scomber scombrus)

5 páginas, 3 tablas, 2 figurasA Real Time-PCR method based on TaqMan technology for the identification of Scomber scombrus has been developed. A system of specific primers and a Minor Groove Binding (MGB) TaqMan probe based on sequences of the mitochondrial cytochrome b region was designed. The method was successfully tested in 81 specimens of S. scombrus and related species and validated in 26 different commercial samples. An average Threshold Cycle (Ct) value of 15.3 was obtained with S. scombrus DNA. With the other species tested fluorescence signal was not detected or Ct was significantly higher (P < 0.001). The efficiency of the assay was estimated to be 92.41%, with 100% specificity, and no cross reactivity was detected with any other species. These results reveal that the developed method is a rapid and efficient tool to unequivocally identify S. scombrus and may aid in the prevention of fraud or mislabelling in mackerel productsThe work was supported by the project “Traceability control mechanisms in the mackerel food chain between Norway and Japan – TraCtrolMac” (No: 251810704011000009)Peer reviewe

Development of a Real-Time PCR method for the identification of Atlantic mackerel (Scomber scombrus)

5 páginas, 3 tablas, 2 figurasA Real Time-PCR method based on TaqMan technology for the identification of Scomber scombrus has been developed. A system of specific primers and a Minor Groove Binding (MGB) TaqMan probe based on sequences of the mitochondrial cytochrome b region was designed. The method was successfully tested in 81 specimens of S. scombrus and related species and validated in 26 different commercial samples. An average Threshold Cycle (Ct) value of 15.3 was obtained with S. scombrus DNA. With the other species tested fluorescence signal was not detected or Ct was significantly higher (P < 0.001). The efficiency of the assay was estimated to be 92.41%, with 100% specificity, and no cross reactivity was detected with any other species. These results reveal that the developed method is a rapid and efficient tool to unequivocally identify S. scombrus and may aid in the prevention of fraud or mislabelling in mackerel productsThe work was supported by the project “Traceability control mechanisms in the mackerel food chain between Norway and Japan – TraCtrolMac” (No: 251810704011000009)Peer reviewe

Identification of Atlantic cod (Gadus morhua), ling (Molva molva), and Alaska pollock (Gadus chalcogrammus) by PCR-ELISA using duplex PCR

8 páginas, 5 figuras, 5 tablasSpecies-specific PCR–ELISA assays for the identification of Atlantic cod (Gadus morhua), Alaska pollock (Gadus chalcogrammus), and ling (Molva molva) in food products have been developed. The method, comprising a set of primers common to the first two species, a set of primers for M. molva, and a probe for each species, was designed using ND4 and cytochrome b genes as molecular markers. The sensitivity and selectivity were then determined for each assay. These assays were afterward used to analyze DNA extracted from commercial fish products. The presence of the target species was successfully detected in all analyzed samples, demonstrating the applicability of this method to the analysis of food products.The work was supported by the projects “ GENTRASEA: Genetic traceability of fish products. Rapid methods with DNA hybridization probes ” and “ LABELFISH: Atlantic network on genetic control of fi sh and seafood labelling and traceability ” . The Spanish Ministry of Science and Innovation is gratefully acknowledged for the doctoral fellowship to L.TPeer reviewe

Developed of a method for the genetic identification of ling species (Genypterus spp.) in seafood products by FINS methodology

5 páginas, 4 figuras, 1 tablaIn the present work a method of authentication of Genypterus and their substitute species was developed, by means of Polymerase Chain Reaction (PCR) technique followed by phylogenetic analysis (FINS, Forensically Informative Nucleotide Sequencing). The methodology developed allows the identification of all the studied species using the mitochondrial cytochrome oxidase subunit I gene (COXI) as molecular marker. Substitutions of the species belonging to Genypterus genera by other species with minor value can take place, since in a lot of seafood products , is not possible the assignation to a particular species based on morphological traits, because it are removed in the transformation process. In this work several methodological strategies were developed and all of them allow the authentication of the studied species in any kind of products, from fresh or frozen fish, to ready-cooked meal. Therefore, the proposed methodology can be used as a routine method to avoid the mislabelling in the marketing of Genypterus species. Also this methodological approximation is suitable to assess the correct seafood traceability of the products elaborated from the mentioned species.Peer reviewe

Supplementary Materials - Exploring the anti-Inflammatory effect of Inulin by integrating transcriptomic and proteomic analyses in a murine macrophage cell model

Supplementary Table S1. Amplification system for the genes of interest and for the genes used for normalization in the qPCR validation. Supplementary Table S2. Normalized counts of all mapped genes from the RNA-Seq assay. Supplementary Table S3. Differentially expressed genes according to the differential expression analysis with DESeq2. Supplementary Table S4. diaPASEF-based protein quantification data for all the samples. A total of 6839 proteins were quantified by diaPASEF LC-MS/MS after processing of the runs as described in the Section 2 and Section 2.5.4. Supplementary Table S5. Differential abundance test for comparison of the LPS + I1 vs. LPS groups. Proteins with CV ≤ 20.0% and quantified in at least 50% of the samples of each group were considered. Fold changes, resulting p-values, and Benjamini–Hochberg corrected p-values for each of the 6065 considered proteins are shown. Supplementary Figure S1. Effect of inulin on the lysosome pathway (KEGG: 04142) from RAW 264.7 LPS-induced inflammation model cells. Pathway diagrams overlayed with the measured protein fold change showing coherent cascades. Differentially expressed genes are represented with positive values in red. Supplementary Figure S2. Effect of inulin on the NF-κB signaling pathway showing integrated transcriptomics data. Genes overexpressed (in red) or underexpressed (green) according to the transcriptomic analysis are highlighted. Supplementary Figure S3. Effect of Inulin on the IL-17 signaling pathway (KEGG: 04657) from RAW 264.7 LPS-induced inflammation model cells. Pathway diagram, overlayed with the measured protein perturbation showing coherent cascades. Differentially expressed genes are represented with negative fold-change values in blue and positive values in red.Peer reviewe

Development of a multiplex PCR-ELISA method for the genetic authentication of Thunnus species and Katsuwonus pelamis in food products

8 páginas, 3 tablas, 3 figurasIn the present work a PCR–ELISA technique for the authentication of Thunnus species was developed. This method is composed by four systems that can be used in a hierarchical way allowing the identification of several scombroids species; or each individual system independently. The hierarchical strategy, proposes a first step, to assign one sample to the Thunnus genus. Next, if the result is positive, several tests can be applied to assign the sample to some particular species of the Thunnus genus. In the case that the result is negative (absence of Thunnus species), it is possible to verify if Katsuwonus pelamis is included in the sample. The method even allows the detection of mixtures of these species in relatively low amounts (up to 1%). Finally, this method was applied to 11 commercial samples to verify the labelling status of tuna products in the market, detecting that 18% were mislabellingThis work was funded by Grant AGL2009-13711-C03-01, Ministerio de Ciencia e Innovación (Spain) in the frame of the project “Trazabilidad Genética de Productos de la Pesca: Métodos rápidos de hibridación de sondas de ADN: GENTRASEA”Peer reviewe