Detection of Fish Allergens in Foods Using an In-House Real-Time PCR Targeting the Ribosomal 18S rRNA Gene.

Fish is one of the major food allergens which, in sensitised individuals, can cause life-threatening allergic reactions, even when present in small amounts. To protect consumers' health, the correct labeling of foods is important. The objective of the present study was to validate an in-house real-time PCR method targeting the ribosomal 18S rRNA gene as universal DNA marker for the detection of fish in foods. The specificity of the primers was assessed on 20 fish species commonly marketed in the Mediterranean basin and other species of molluscs and crustaceans and foods of animal and plant origin. The absolute detection of the method was assessed using DNA extracted from a fish mixture and the SureFood® QUANTARD Allergen 40 reference material. The relative amount was assessed on a fish and béchamel sauce blend. Commercial food samples either labelled with or without fish in the ingredient list, were tested for the presence of fish DNA. The primer showed high specificity against the selected fish species. The limit of detection (LOD) and limit of quantification (LOQ) of the in-house method were 0.5 pg/µL and 5 pg/µL, respectively. The relative quantification in fish and béchamel blend samples detected a concentration as low as 0.000025%, corresponding to 0.25 mg/kg of fish, indicating the suitability of the method in a food matrix. The presence of fish DNA was always detected in commercial samples in which the presence of fish was listed in the ingredient list. The method was able to detect the presence of fish DNA also in samples in which the presence of fish was indicated as traces or was not declared on the label. The proposed method was demonstrated to be a reliable, specific, and sensitive method for the detection of fish allergens in foods. Therefore, the proposed real-time PCR method could be used as a useful instrument in the verification of compliance with allergen labelling regulations.

Determination of phytoplankton in water samples, algal biotoxins, microbiological parameters and microplastics in Mediterranean mussels (Mytilus galloprovincialis Lamarck, 1819) from an experimental pilot farm in the Calich Lagoon (Sardinia, Italy).

The aims of this paper were to collect and analyse preliminary data of phytoplankton in the water, biotoxins, Escherichia coli, Salmonella spp., Vibrio spp. and microplastic eventually present in farmed mussels, and to acquire information about the production capability from an experimental pilot farm of the Calich Lagoon. Two sampling sessions were carried out, in February and in May 2019, also monitoring the water condition (pH, temperature, salinity, dissolved oxygen, chlorophyll a). No potentially toxic algae were detected, and moreover no biotoxins (Paralytic Shellfish Poison, Diarrheic Shellfish Poison, Amnesic Shellfish Poison) were found in mussels. E.coli was present with the highest concentration in February (16000 MPN/100g e.p.). Salmonella and Vibrio spp. have not been detected. Almost a microplastic per grams was found, mainly fiber of different colours. Further studies, carried out for several months, will allow to better understand the possible problems related to the production of mussels in a lagoon not yet classified as a shellfish production area.

Identification and quantification of potential microplastics in shellfish harvested in Sardinia (Italy) by using transillumination stereomicroscopy.

Plastics are non-biodegradable polymers made up of different groups of petrochemical materials. Several biotic and abiotic factors can change the density of plastic fragmenting it and originating microplastics (MPs). MPs have been defined as small pieces of plastic less than 5 mm in size. Due to their small size, they are an emerging concern in the marine environment since they can be ingested by aquatic organisms, especially filter-feeding organisms, such as bivalve mollusks. Impacts of MPs exposure have been shown at various levels of biological organization, from cellular to tissue to individual and population levels. For example, oxidative stress and inflammation have been observed in copepods and mussels, obstruction and physical damage of the digestive tract were found in fish and swimming behavior alterations, disruption of foraging and feeding behavior and overall reduced fitness and survival were observed in fish and oysters. In addition, MPs can act as a vector for the transfer of chemicals to marine biota. The aim of the present study was the identification and quantification of potential MPs in shellfish harvested in Sardinia (Italy) by using transillumination stereomicroscopy. Bivalves were collected from 4 of the main production areas located along the Sardinian coast and selected according to the principles of the risk assessment. The results of the present study demonstrated the presence of potential MPs in 70% of the analyzed samples: the presence of MPs in bivalve mollusks may pose a threat to food safety, and there is an urgent need to evaluate the potential risks of MPs to human health.

Influence of seasonality on the presence of okadaic acid associated with Dinophysis species: A four-year study in Sardinia (Italy).

In Sardinia (Italy), bivalve molluscs production plays an important role in the trade balance. Diarrhoetic shellfish poisoning (DSP), an intoxication caused by the ingestion of bivalve molluscs that have accumulated high levels of Okadaic acid (OA), may represent a serious risk for the public health and a remarkable economic loss for the producers. Aim of this work was to improve knowledge about the repeatability of OA accumulation phenomena in various seasons trying to understand whether or not there was a trend. Also, the interaction between toxic algae and OA accumulation was examined. In this study, data of lipophilic toxins, water temperature and abundance of DSP-producing microalgal species were collected in a four-year period (2015-2018) in coastal production areas of Sardinia. Several episodes of OA positive values (>160 eq µgAO/Kg pe, Reg 853/04) were recorded during the study period in different production areas of Sardinia and in different seasons. A seasonal repeatability of OA accumulation in molluscs was observed in some production areas; moreover, different temporal gaps between the presence of toxic algae and OA accumulation were reported. Toxicity was observed almost exclusively in Mytilus galloprovincialis Lamark (99%), being this matrix the most abundant species bred in Sardinia.

New evidence of pectenotoxins in farmed bivalve molluscs from Sardinia (Italy).

Several planktonic dinoflagellates can produce lipophilic phycotoxins that represent a significant threat to public health as well as to shellfish and fish farming. Poisoning related to some of these toxins is categorised as diarrhetic shellfish poisoning. We analysed 975 shellfish samples from Tortoli in the central-eastern region of Sardinia (Italy) from January 2016 to March 2020, to investigate the prevalence of different lipophilic marine biotoxins in mollusc bivalves. The results highlighted the predominant presence of toxins belonging to the okadaic acid group in all samples with toxin concentrations exceeding legal limits, and revealed the new occurrence of pectenotoxins in oysters and clams with a winter seasonality in recent years. The origin of shellfish toxicity was associated with the same Dinophysis species, mainly D. acuminata. Based on both these results and other precedents, monitoring and recording systems are strongly recommended.

Development of a Digital RT-PCR Method for Absolute Quantification of Bluetongue Virus in Field Samples.

Bluetongue (BT) is a major Office International des Epizooties (OIE)-listed disease of wild and domestic ruminants caused by several serotypes of Bluetongue virus (BTV), a virus with a segmented dsRNA genome belonging to the family Reoviridae, genus Orbivirus. BTV is transmitted through the bites of Culicoides midges. The aim of this study was to develop a new method for quantification of BTV Seg-10 by droplet digital RT-PCR (RTdd-PCR), using nucleic acids purified from complex matrices such as blood, tissues, and midges, that notoriously contain strong PCR inhibitors. First, RTdd-PCR was optimized by using RNAs purified from serially 10-fold dilutions of a BTV-1 isolate (105.43TCID50/ml up to 10-0.57 TCID50/ml) and from the same dilutions spiked into fresh ovine EDTA-blood and spleen homogenate. The method showed a good degree of linearity (R 2 ≥ 0.995). The limit of detection (LoD) and the limit of quantification (LoQ) established were 10-0.67TCID50/ml (0.72 copies/µl) and 100.03TCID50/ml (3.05 copies/µl) of BTV-1, respectively. Second, the newly developed test was compared, using the same set of biological samples, to the quantitative RT-PCR (RT-qPCR) detecting Seg-10 assay widely used for the molecular diagnosis of BTV from field samples. Results showed a difference mean of 0.30 log between the two assays with these samples (p < 0.05). Anyway, the analysis of correlation demonstrated that both assays provided similar measurements with a very close agreement between the systems.

Detection of fish allergen by droplet digital PCR.

Fish is one of fourteen allergens that must be highlighted on the label within the ingredients list. The European regulation is very restrictive to allergens with zero tolerance. Therefore, it is important to establish sensitive and specific methods for detecting fish allergen. Applicability to detect and quantify fish allergen by droplet digital polymerase chain reaction (ddPCR) has been evaluated in this work. Genomic DNA of three species belonging to the most common fish families were analyzed. PCR primers were designed to amplify a 166 bp region of the 18S rRNA gene. Comparative studies were performed to establish the optimal primer and probe concentrations. Annealing temperature was determined by using thermal gradient. The results have shown good applicability of the optimized 18S rRNA gene-method to detect and quantify small amounts of the target in samples analyzed. However, validation studies are needed in order to apply ddPCR technology for routine allergens analysis.

Detection of Dinophysis Species and Associated Okadaic Acid in Farmed Shellfish: a Two-year Study from The Western Mediterranean Area.

INTRODUCTION: Diarrhoetic shellfish poisoning (DSP), an alimentary intoxication known to lead to intestinal symptoms, and caused by toxins produced by some dinoflagellates (including several Dinophysis), represents a serious threat to public health. The aim of this paper was to provide information about the occurrence and abundance of potentially toxic harmful algal species causing DSP, and the associated concentration of okadaic acid (OA) toxins. The departing assumption was that in the study area there was an increase in the presence both of Dinophysis species and OA and its derivates that could result in a risk to the health of seafood consumers. MATERIAL AND METHODS: During 2015-2016, water and shellfish samples were collected in the Mediterranean area (Sardinia, Italy). Dinophysis cells were counted according to Utermöhl's method from water samples, while mass spectrometry was used to identify lipophilic toxins in molluscs. RESULTS: A total of 46 non-compliant samples of Mytilus galloprovincialis were observed. Their non-compliance concerned their OA levels above the legal limit. Among toxic dinoflagellates, D. acuminata and D. sacculus were the species found mostly during DSP events. CONCLUSION: No cases of human intoxication have been reported, but continuous surveillance of toxic phytoplankton is necessary to predict and prevent its harmful effects on human health.

Analysis of Foreign Matter in Foodstuffs Using the Light Filth Test: Report 2012-2013.

The European legislation set the new hygiene standards in food chain with the purpose to ensure high levels of public health protection in relation to food production. In order to guarantee excellent hygiene standards in food chain, particular attention must be paid to the presence of foreign matter, like light solid impurities of mineral, vegetable or animal origin. The light filth test is a suitable method used to detect and count light solid impurities applicable to different foodstuffs. We report the results of the analysis of 93 foodstuffs official samples investigated for the presence of foreign matter at the Institute for Experimental Veterinary Medicine of Sardinia, from 2012 to 2013. Insect fragments were found in a sample of semolina and in a sample of canned tomato; plastic fragments were found in a sample of grated bread.

Foreign Bodies in Dried Mushrooms Marketed in Italy.

The presence of foreign bodies in mushrooms affects their marketability and may result in health risks to consumers. The inspection of fresh or dried mushrooms today is very important in view of the increased consumption of this kind of food. Ten samples of dried mushrooms collected in supermarkets were examined for evidence of entomological contamination by macro and microscopic analytical methods, the so-called filth-test. A total of 49 46 determinations, comprising 15 g of the vegetable matrix, were made. The microscopic filth test consistently detected an irregular distribution of physical contaminants following repeated determinations of the same sample. Visual examination, on the other hand, was not sufficient to ensure a product free of contaminants.

Monitoring the Presence of Genetically Modified Potato EH92-527-1 (BPS-25271-9) in Commercial Processed Food.

The Amflora (EH92-527-1) potato is a genetically modified (GM) potato in which only starch of the amylopectin form is produced. This has been achieved by intervening with the biosynthesis of starch in this variety of potato. The Amflora potato is solely grown for the purposes of enhancing its industrial application. Although the Amflora potato is not fit for human consumption, the presence of the potato itself or any of its derived products in the food chain cannot be excluded, it should be considerate adventitious or technically unavoidable and can be accepted in a proportion no higher than 0.9%. To achieve the goal of our work we analysed forty-five potato-derived products to evaluate transgenic potato presence by real time polymerase chain reaction, obtaining negative results. In order to verify the correct application of the law and to assure the quality for the consumer, it is necessary to continue GM monitoring to verify the adventitious presence itself in food.