FAST Real Time PCR for control of intra-species recycling in aquaculture feed, focused to the most relevant fish species farmed in Europe.

Recent regulations in animal feed composition prohibit intra-species recycling, the recycling of one given animal species to the same species, in order to avoid potential safety risks to human and animal health. These regulations have generated the need of their control in aquaculture by effective and specific analytical techniques. To date, most studies of species identification and detection in feedstuffs are focused on land species, but few studies are focused on species composition in fish feed. The present work describes five methodologies based in Real Time PCR for detection of the most relevant fish species farmed in Europe: gilthead sea bream (Sparus aurata); sea bass (Dicentrarchus labrax); turbot (Scophthalmus maximus); rainbow trout (Onchorynchus mykiss); and salmon (Salmo salar), in order to guarantee the intra-species recycling regulation in aquaculture feedstuffs.

Effect of the industrial canning on the toxicity of mussels contaminated with diarrhetic shellfish poisoning (DSP) toxins.

The effect of canning in pickled sauce and autoclaving on weight, toxin content, toxin concentration and toxicity of steamed mussels was studied. Weight decreased by 25.5%. Okadaic acid (OA) and DTX2 content of mussel meat decreased by 24.1 and 42.5%, respectively. The estimated toxicity of the mussel remained nearly unchanged (increased by 2.9%). A part of the toxins lost by the mussels was leached to the sauce but the remaining part should have been thermally degraded. DTX2 underwent more degradation than OA and, in both toxins, free forms more than conjugated ones. This process, therefore, cannot be responsible for the large increments of toxicity of processed mussels -relative to the raw ones-sometimes detected by food processing companies. The final product could be monitored in several ways, but analysing the whole can content or the mussel meat once rehydrated seems to be the most equivalents to the raw mussel controls.

Effect of the industrial steaming on the toxicity, estimated by LC-MS/MS, of mussels exposed for a long time to diarrhetic shellfish poisoning (DSP) toxins.

The effect of industrial steaming on mussels that had been naturally exposed to DSP toxins for a long time was studied using LC-MS/MS. The estimated toxicity increased with steaming by a percentage that cannot be explained by weight loss. The estimated toxin content per mussel increased substantially with the treatment, which can only be explained by an incorrect estimation by the technique (at the extraction or analytical level) or by the presence of unknown derivatives or analogues. Direct alkaline hydrolysis of the mussel meat yielded more toxin than the standard hydrolysis (hydrolysis of the methanolic extracts), suggesting that extraction was, at least in part, responsible for the increase of toxin content. In situations as the one described in this work, it can be expected that mussels with toxicities well below the regulatory limit could easily surpass that level after industrial steaming, thus producing important losses for food processors.

lolB gene, a valid alternative for qPCR detection of Vibrio cholerae in food and environmental samples.

In recent years a new genetic target for Vibrio cholerae detection has been reported, but its application was limited to clinical samples. This target, lolB, has never been applied to either food or environmental samples. In the present study the development, as well as the evaluation and pre-validation, of a real-time PCR method based on lolB, is described. The method included a newly designed hydrolysis probe to enhance its specificity. After comparison against other molecular and traditional methods, similar results were obtained regarding relative sensitivity, relative specificity, relative accuracy, positive and negative predictive values and index kappa of concordance (all higher than 91%), as well as a very low limit of detection (2 cfu/25 g). Additionally, after the analysis of more than 160 different food and environmental samples, its applicability in the food industry was completely demonstrated.

Detection of the causal agents of Keriorrhea (Lepidocybium flavobrunneum and Ruvettus pretiosus) by means of Real Time PCR.

Consumption of Lepidocybium flavobrunneum and Ruvettus pretiosus is related with the gastrointestinal disease called Keriorrhea. Sometimes, intentionally or not, these species are mislabelled as other harmless species, causing severe disruptions to consumers. The correct identification of these species helps to avoid food fraud and health problems. For this reason, a multiplex Real Time-PCR method based on TaqMan technology for the correct authentication of L. flavobrunneum and R. pretiosus has been developed. The method is based on a species-specific set of primers and TaqMan probe which amplifies a 276 bp fragment of the cytochrome oxidase I (COI) mitochondrial DNA region. This methodology allows your application to any type of product, regardless of the degree of processing it has undergone with high specificity, sensitivity and rapidity. Also, it might be a useful tool in monitoring and verifying labelling regulation and protect consumer rights.

Development of an in-house fast real-time PCR method for detection of fish allergen in foods and comparison with a commercial kit.

Food allergy is recognised as an important human health problem. Fish represent one of the most important causes of food hypersensitivity reaction. Small amounts of the allergen can cause severe reactions in sensitive individuals, so correct labelling is essential to ensure the protection of consumers. The objective of the present work was to develop a reliable, sensitive and specific real-time PCR method for the detection of fish and traces of fish in all kind of products included those that have undergone aggressive treatments such as high temperature or pressure. This methodology was validated simulating products likely to contain this allergen and spiking them with fish cooking water. In addition, a comparison between the performance of in-house methodology and a commercial kit, both of them based on real-time PCR, was carried out. This work is relevant because it is the first, rapid real-time PCR method developed to date for the detection of fish in processed food products. The results obtained confirm the present assay is a useful tool in detecting fish and, therefore, minimising exposure and reducing incidences of allergic reaction to fish in contaminated products.

In-house validation of a multiplex real-time PCR method for simultaneous detection of Salmonella spp., Escherichia coli O157 and Listeria monocytogenes.

A wide variety of qPCR methods currently exist for Salmonella spp., Escherichia coli O157 and Listeria monocytogenes detection. These methods target several genes and use different detection chemistries, either in simplex or in multiplex formats. However, the majority of these methods have not been carefully validated, and the number of validated methods that use multiplex qPCR is even lower. The aim of the present study was to develop and validate a multiplex qPCR method from previously validated simplex qPCR primers and probes. A modified broth medium was selected and primary and secondary enrichment times were further optimized. Efficiency of the newly combined qPCR system was comprised between 91% and 108%, for simplex and multiplex analyses. A total of 152 food and environmental, natural and spiked samples, were analyzed for the evaluation of the method obtaining values above 91% that were reached for all the quality parameters analyzed. A very low limit of detection (5 cfu/25 g after enrichment) for simultaneous identification of these 3 pathogens was obtained.

Quantitative determination of the okadaic acid toxins group by a colorimetric phosphatase inhibition assay: interlaboratory study.

An interlaboratory collaborative study to validate a colorimetric phosphatase inhibition assay for quantitative determination of the okadaic acid (OA) toxins group in molluscs, OkaTest, was conducted. Eight test materials, including mussels, scallops, clams, and cockles, were analyzed as blind duplicates. Blank samples and materials containing different OA toxin levels ranging from 98 to 275 microg/kg OA equivalents were included. The study was carried out by a total of 16 laboratories from 11 different countries. Values obtained for repeatability relative standard deviations (RSDr) ranged from 5.4 to 11.2% (mean 7.5%). Reproducibility RSD (RSD(R)) values were between 7.6 and 13.2% (mean 9.9%). The Horwitz ratio (HorRat) values ranged between 0.4 and 0.6. A recovery assay was also carried out using a sample spiked with OA. A mean recovery of 98.0% and an RSD of 14.5% were obtained. The results obtained in this validation study indicate that the colorimetric phosphatase inhibition assay, OkaTest, is suitable for quantitative determination of the OA toxins group. OkaTest could be used as a test that is complementary to the reference method for monitoring the OA toxins group.

In vitro approaches to evaluate toxicity induced by organotin compounds tributyltin (TBT), dibutyltin (DBT), and monobutyltin (MBT) in neuroblastoma cells.

The toxic effects of the organotin compounds (OTCs) monobutyltin (MBT), dibutyltin (DBT), and tributyltin (TBT) were evaluated in vitro in a neuroblastoma human cell line. Mechanisms of cell death, apoptosis versus necrosis, were studied by using several markers: inhibition of cell viability and proliferation, F-actin, and mitochondrial membrane potential changes as well as reactive oxygen species (ROS) production and DNA fragmentation. The most toxic effects were detected with DBT and TBT even at very low concentrations (0.1-1 µM). In contrast, MBT induced lighter cytotoxic changes at the higher doses tested. None of the studied compounds stimulated propidium iodide uptake, although the most toxic chemical, TBT, caused lactate dehydrogenase release at the higher concentrations tested. These findings suggest that in neuroblastoma, OTC-induced cytotoxicity involves different pathways depending on the compound, concentration, and incubation time. A screening method for DBT and TBT quantification based on cell viability loss was developed, allowing a fast detection alternative to complex methodology.

Authentication of male beef by multiplex fast real-time PCR.

Meat is a significant source of high-quality nutrients, which are very important in the diet. Among meat products, one of the most prized is bovine meat, of which male beef has been designated to be of a higher quality. However, because of its similarity with female beef, deliberate or unintentional substitutions can occur. To avoid this, methodology based on the fast real-time polymerase chain reaction has been developed to authenticate the species and gender origin of beef. This technique consists of two polymerase chain reactions: one bovine-specific reaction and another Y-chromosome-specific multiplex reaction. This methodology has been validated for all kinds of beef products, including those subjected to intensive processing treatments, and it has subsequently been applied to 10 commercial samples labelled as ox to determine whether they are properly labelled. This assay has been shown to have high specificity, sensitivity and rapidity, with the potential to be a powerful tool in enforcing food labelling regulations.

Fast Real-Time PCR assay for detection of Tetramicra brevifilum in cultured turbot.

Global aquaculture production of turbot has rapidly increased worldwide in the last decade and it is expected to have even bigger growth in the next years due to new farms operating. The losses caused by pathogen infections have grown at the same time as the production of this species. Parasitological infections are among the main relevant pathologies associated with its culture and produce serious losses in aquaculture, reduce the growth rate in fish and may lead to unmarketable fish due to skeletal muscle abnormalities in cases with high intensity of infection. The microsporidian parasite Tetramicra brevifilum causes severe infections and generates major losses in farmed turbot. Infections are difficult to control due to spore longevity and its direct transmission. To facilitate the infection management, an effective tool for fast detection and identification of T. brevifilum is needed. This study provides a molecular methodology of fast Real-Time PCR for T. brevifilum detection to the aquaculture industry, useful for routine control of T. brevifilum at turbot farms. The method is characterized by its high specificity and sensitivity, and it can be applied to cultured turbot for parasite detection regardless of the life-cycle stage of the pathogen or the infection intensity.

Rapid method for controlling the correct labeling of products containing common octopus (Octopus vulgaris) and main substitute species (Eledone cirrhosa and Dosidicus gigas) by fast real-time PCR.

The TaqMan real-time PCR has the highest potential for automation, therefore representing the currently most suitable method for screening, allowing the detection of fraudulent or unintentional mislabeling of species. This work describes the development of a real-time polymerase chain reaction (RT-PCR) system for the detection and identification of common octopus (Octopus vulgaris) and main substitute species (Eledone cirrhosa and Dosidicus gigas). This technique is notable for the combination of simplicity, speed, sensitivity and specificity in an homogeneous assay. The method can be applied to all kinds of products; fresh, frozen and processed, including those undergoing intensive processes of transformation. This methodology was validated to check how the degree of food processing affects the method and the detection of each species. Moreover, it was applied to 34 commercial samples to evaluate the labeling of products made from them. The methodology herein developed is useful to check the fulfillment of labeling regulations for seafood products and to verify traceability in commercial trade and for fisheries control.

Fast real-time PCR for the detection of crustacean allergen in foods.

Crustaceans are one of the most common allergens causing severe food reaction. These food allergens are a health problem, and they have become very important; there are various regulations that establish that labeling must be present regarding these allergens to warn consumers. In the present work a fast real-time PCR, by a LNA probe, was developed. This allows the detection of crustaceans in all kinds of products, including processed products in which very aggressive treatments of temperature and pressure during the manufacturing process are used. This methodology provides greater sensitivity and specificity and reduces the analysis time of real-time PCR to 40 min. This methodology was further validated by means of simulating products likely to contain this allergen. For this, products present on the market were spiked with crustacean cooking water. The assay is a potential tool in issues related to the labeling of products and food security to protect the allergic consumer.

Real-time PCR method applied to seafood products for authentication of European sole (Solea solea) and differentiation of common substitute species.

Judged by quality and taste, the European sole (Solea solea) is considered one of the finest flatfish and is, thus, of considerable commercial value. In the present work, a specific fast real-time PCR was developed for the authentication of S. solea, i.e. to distinguish it from other related species and avoid substitution of this species, either deliberately or unintentionally. The method is based on a species-specific set of primers and MGB Taqman probe which amplifies a 116-bp fragment of the internal transcribed spacer 1 (ITS 1) ribosomal DNA region. This assay combines the high specificity and sensitivity of real-time PCR with the rapidity of the fast mode, allowing the detection of S. solea in a short period of time. The present methodology was validated for application to all types of manufactured products for the presence of S. solea, with successful results. Subsequently, the method was applied to 40 commercial samples to determine whether correct labeling had been employed in the market. It was demonstrated that the assay is a useful tool in monitoring and verifying food labeling regulations.

Cytotoxic activity of extracts of marine sponges from NW Spain on a neuroblastoma cell line.

Six species of marine sponges collected at intertidal and sublittoral sites of the coast of Galicia (NW Spain) were screened for potential cytotoxic properties on Neuroblastoma BE(2)-M17 cell line. Exposure to Halichondria panicea, Pachymatisma johnstonia, Ophlitaspongia seriata and Haliclona sp. aqueous extracts strongly affected cell appearance, inducing loss of neuron-like morphology and the formation of clumps. Extracts from these species also caused significant rates of cell detachment and decrease of mitochondrial membrane potential. Incubation with P. johnstonia, O. seriata and Suberites massa extracts also decreased the rate of cell proliferation. The increase of incubation time enhanced propidium iodide uptake by neuroblastoma cells. Toxic responses triggered by sponge extracts are compatible with apoptotic phenomena in neuroblastoma cells, even though increasing propidium uptake at long periods of exposure might indicate the induction of secondary necrosis. The cytotoxic properties of the tested extracts suggest the presence of compounds with potential pharmacological or biotechnological applications in the screened sponge species.

Dietary uptake of polybrominated diphenyl ethers (PBDEs), occurrence and profiles, in aquacultured turbots (Psetta maxima) from Galicia, Spain.

Polybromodiphenyl ethers (PBDEs) are one of the many toxic chemicals present in the environment and in the food we eat every day, being fish one of the main sources of persistent organic pollutants in our diet; like other lipid-related contaminants, they are of concern since they can bioaccumulate and biomagnify through the trophic chain. We published a study focused on the dietary uptake of dioxins and furans (PCDD/Fs) and dioxin-like polychlorobiphenyls (dl-PCBs) in a set of samples of Spanish farmed turbot (Blanco et al., 2007). In the present paper, we extend the study to PBDEs to provide more information about the uptake and transfer from feed to fish of halogenated contaminants. PBDEs in the feeds (2.35-4.76 ng g(-1)) were reflected in turbot fillets (0.54-2.05 ng g(-1)): predominant congeners were tetra-BDE 47, penta-BDEs 99 and 100. It is remarkable that tetra-BDE 49, accounting for only 2% in the feed, contributed to 15% of total PBDEs in turbot fillets. Dietary net accumulation values, 30-45%, showed that tri-, tetra-, penta- and hexa-BDEs were as efficiently transferred into turbot as dl-PCBs and tetra- and penta-chlorinated PCDD/Fs. Lipid-normalized biomagnification factors relating concentration in fish and in feed, BMFs>1 were obtained, except for BDE 209. BDE 49 accumulation, 90%, was possibly contributed by metabolism of higher brominated BDEs. Implication in aquaculture management is a need for uncontaminated fish feed to offer safe products.

Cyclic imines: chemistry and mechanism of action: a review.

In recent years, there has been an increase in the production of shellfish and in global demand for seafood as nutritious and healthy food. Unfortunately, a significant number of incidences of shellfish poisoning occur worldwide, and microalgae that produce phycotoxins are responsible for most of these. Phycotoxins include several groups of small to medium sized natural products with molecular masses ranging from 300 to over 3000 Da. Cyclic imines (CIs) are a recently discovered group of marine biotoxins characterized by their fast acting toxicity, inducing a characteristic rapid death in the intraperitoneal mouse bioassay. These toxins are macrocyclic compounds with imine (carbon-nitrogen double bond) and spiro-linked ether moieties. They are grouped together due to the imino group functioning as their common pharmacore and due to the similarities in their intraperitoneal toxicity in mice. Spirolides (SPXs) are the largest group of CIs cyclic imines that together with gymnodimines (GYMs) are best characterized. Although the amount of cyclic imines in shellfish is not regulated and these substances have not been categorically linked to human intoxication, they trigger high intraperitoneal toxicity in rodents. In this review, the corresponding chemical structures of each member of the CIs and their derivatives are reviewed as well as all the data accumulated on their mechanism of action at cellular level.

Genetic identification of horse mackerel and related species in seafood products by means of forensically informative nucleotide sequencing methodology.

In the present study, a methodology based on the amplification of a fragment of mitochondrial cytochrome b and subsequent phylogenetic analysis (FINS: forensically informative nucleotide sequencing) to genetically identify horse mackerels have been developed. This methodology makes possible the identification of more than 20 species belonging to the families Carangidae, Mullidae, and Scombridae. The main novelty of this work lies in the longest number of different horse mackerel species included and in the applicability of the developed methods to all kinds of processed products that can be found by consumers in markets around the world, including those that have undergone intensive processes of transformation, as for instance canned foods. Finally, the methods were applied to 15 commercial samples, all of them canned products. Therefore, these methods are useful for checking the fulfillment of labeling regulations for horse mackerels and horse mackerel products, verifying the correct traceability in commercial trade, and fisheries control.

Authentication of Atlantic salmon (Salmo salar) using real-time PCR.

A real-time PCR assay based on LNA TaqMan probe technology was developed for the detection and identification of Atlantic salmon (Salmo salar). Among the advantages it is worth highlighting simplicity, rapidity, highest potential for automation and minor risk of contamination of this technique. The TaqMan real-time PCR is the currently most suitable method for screening, allowing the detection of fraudulent or unintentional mislabelling of this species. The method can be applied to all kind of products, fresh, frozen and processed products, including those undergoing intensive processes of transformation. The developed methodology using specific primers-probe set was validated and further applied to 20 commercial samples labelled as salmon or S. salar in order to determinate if the species used for their manufacturing corresponded to this species. The methodology herein developed is useful to check the fulfilment of labelling regulations for seafood products, verify the correct traceability in commercial trade and for fisheries control.

Whey protein-based coatings on frozen Atlantic salmon (Salmo salar): Influence of the plasticiser and the moment of coating on quality preservation.

The effects of different whey protein concentrate coating formulations (with or without glycerol or sorbitol in two proportions) on frozen Atlantic salmon quality parameters were evaluated. The influence of the moment of coating application (before or after freezing) was also studied. The coating application after freezing increased the thaw yield, decreased the drip loss, and modified colour parameters of frozen and thawed fillets, in comparison with application before freezing. The moment of coating also influenced the colour of cooked fish fillets. The type of plasticiser affects the colour of thawed and cooked samples, but not the colour of frozen samples. The protein coatings delayed lipid oxidation of salmon fillets, providing better protection against it than water glazing, and this effect was more pronounced when glycerol instead of sorbitol was used in the coating formulation. WPC+glycerol (1:1) coating was the best for frozen Atlantic salmon protection. The sensory properties of salmon fillets were not modified by the use of this coating.