The world's seafood supply and trade have increased in the last decades, as well as the potential for marketed species substitution. Currently, seafood safety and authenticity assessment have become ...central issues, directly related with the identification of improper labeling of processed foods. To detect and prevent mislabeling issues, species identification using DNA barcodes has been widely used as effective molecular markers. Therefore, this review intends to present the current status on the application of DNA barcodes to seafood species authentication. In this regard, the barcode regions, reference databases and related methodologies are described, while applications are listed and summarized. Cytochrome c oxidase subunit I (COI) gene has been the preferential targeted DNA region in animal species identification, including fish and shellfish, though other mitochondrial (cytb, 12S rRNA, 16S rRNA) and nuclear genes have been used. DNA barcoding relying on Sanger's sequencing has been the most used approach for seafood authentication. Nevertheless, in recent years, noteworthy progresses have been advanced toward DNA barcoding strategies, involving next generation sequencing. Methods relying on real-time PCR using species-specific primers and probes or followed by high resolution melting analysis combined with DNA barcodes represent alternative and promising approaches for simple, cost-effective and high-throughput species discrimination in processed seafood. Still, polymerase chain reaction with restriction fragment length polymorphism detection, targeting DNA barcodes, continues to be a well-established and broadly accepted method in seafood authentication.
•DNA barcoding was combined with High Resolution Melting (HRM) analysis.•COI and cytb barcode regions were exploited for Gadidae species differentiation.•Atlantic cod, Pacific cod, pollock and saithe ...were identified successfully.•Cytb was the most efficient barcode to differentiate the four species by HRM analysis.•A new, rapid, simple and cost-effective tool was proposed for codfish authentication.
This work aimed to exploit the use of DNA mini-barcodes combined with high resolution melting (HRM) for the authentication of gadoid species: Atlantic cod (Gadus morhua), Pacific cod (Gadus macrocephalus), Alaska pollock (Theragra chalcogramma) and saithe (Pollachius virens). Two DNA barcode regions, namely cytochrome c oxidase subunit I (COI) and cytochrome b (cytb), were analysed in silico to identify genetic variability among the four species and used, subsequently, to develop a real-time PCR method coupled with HRM analysis. The cytb mini-barcode enabled best discrimination of the target species with a high level of confidence (99.3%). The approach was applied successfully to identify gadoid species in 30 fish-containing foods, 30% of which were not as declared on the label. Herein, a novel approach for rapid, simple and cost-effective discrimination/clustering, as a tool to authenticate Gadidae fish species, according to their genetic relationship, is proposed.
•Universal primers targeting the 16S rRNA gene were designed for fish detection.•Two real-time PCR systems based on the EvaGreen dye and a TaqMan probe were compared.•Sensitivities of 0.01 pg of fish ...DNA and 0.0001% of fish in béchamel were reached.•The probe system was successfully validated in terms of precision and trueness.•A high level of mislabelling for fish as allergen was verified in foodstuffs.
Fish is one of the most common allergenic foods that should be accurately labelled to protect the health of allergic consumers. In this work, two real-time PCR systems based on the EvaGreen dye and a TaqMan probe are proposed and compared. New primers were designed to target the 16S rRNA gene, as a universal maker for fish detection, with fully demonstrated specificity for a wide range of fish species. Both systems showed similar absolute sensitivities, down to 0.01 pg of fish DNA, and adequate real-time PCR performance parameters. The probe system showed higher relative sensitivity and dynamic range (0.0001–50%) than the EvaGreen (0.05–50%). They were both precise, but trueness was compromised at the highest tested level with the EvaGreen assay. Therefore, both systems were successful, although the probe one exhibited the best performance. Its application to verify labelling compliance of foodstuffs suggested a high level of mislabelling and/or fraudulent practices.
► Traceability of transgenic maize in a traditional bread. ► Effect of breadmaking on DNA degradation. ► Effect of breadmaking on GMO quantification. ► Effect of raw material used in breadmaking on ...GMO quantification.
Broa is a maize bread highly consumed and appreciated, especially in the north and central zones of Portugal. In the manufacturing of broa, maize flour and maize semolina might be used, besides other cereals such as wheat and rye. Considering the needs for genetically modified organism (GMO) traceability in highly processed foods, the aim of this work was to assess DNA degradation, DNA amplification and GMO quantification along breadmaking process of broa. DNA degradation was noticed by its decrease of integrity after dough baking and in all parts of bread sampling. The PCR amplification results of extracted DNA from the three distinct maize breads (broa 1, 2 and 3) showed that sequences for maize invertase gene and for events MON810 and TC1507 were easily detected with strong products. Real-time PCR revealed that quantification of GMO was feasible in the three different breads and that sampling location of baked bread might have a limited influence since the average quantitative results of both events after baking were very close to the actual values in the case of broa 1 (prepared with maize semolina). In the other two maize breads subjected to the same baking treatment, the contents of MON810 maize were considerably underestimated, leading to the conclusion that heat-processing was not the responsible parameter for that distortion, but the size of particle and mechanical processing of raw maize play also a major role in GMO quantification.
Maize, the second most important genetically modified (GM) crop, has the highest number of authorised GM events for food and feed in the EU. To provide consumer's information, labelling for food ...products containing more than 0.9% of GM material is demanded by the actual EU legislation. Analysis of foods is then essential to detect and quantify GM maize material and verify the compliance with labelling information. The aim of the present work was to assess the presence of GM maize in a range of processed foods commercialised in Portugal between 2007 and 2010. For this purpose, screening of GM material was carried out by qualitative PCR targeting the 35S promoter and the NOS terminator, followed by the specific detection of Bt11, MON810, Bt176, GA21, MON863, NK603, TC1507 (also known as DAS1507), DAS59122 and MIR604 events. The identified maize events were confirmed and quantified by real-time PCR with hydrolysis probes. The overall results of GMO screening were 30% for 35S promoter, 10% for NOS terminator and 25% for identified events. The most frequently detected events were MON810, TC1507 and NK603, with one sample containing GA21, while the other events were not detected in any of the analysed foods. The quantitative results suggest the need for a more severe control since 4% of the analysed foods contained more than the threshold for labelling and none of them declared the presence of GMO.
•Prevalence of GMO in processed foods commercialised in Portugal from 2007 to 2010.•Detection of Bt11, MON810, Bt176, GA21, MON863, NK603, TC1507, DAS59122, MIR604 maize.•TaqMan real-time PCR quantification of MON810, TC1507, NK603 and GA21 events.•4% of the Analysed foods had more than 0.9% GM maize.
Gadiform order includes several fish families, from which Gadidae and Merlucciidae are part of, comprising the most commercially important and highly appreciated fish species, such as cod, pollock, ...haddock, and hake. Parvalbumins, classified as calcium-binding proteins, are considered the main components involved in the majority of fish allergies. Nine and thirteen parvalbumins were identified in different fish species from Gadidae and Merlucciidae families, respectively. This review intends to describe their molecular characterization and the clinical relevance, as well as the prevalence of fish allergy. In addition, the main protein- and DNA-based methods to detect fish allergens are fully reviewed owing to their importance in the safeguard of sensitized/allergic individuals.
The consumption of plant food supplements (PFS) has been growing globally, with an increase of misleading labeling and fraudulent practices also being reported. Recently, the use of molecular biology ...techniques has been proposed to detect botanical adulterations, one of the possible frauds in PFS. However, difficulties in recovering DNA from some PFS samples have been described. Aiming at using DNA-based methods for the unequivocal identification of plant species in PFS, adequate DNA isolation is required. However, PFS often contain pharmaceutical excipients known to have adsorbent properties that might interfere with DNA extraction. Thus, the aim of this work was to assess the effect of different excipients (talc, silica, iron oxide and titanium dioxide) on the recovery/amplification of DNA. For that purpose, known amounts of template maize DNA were spiked either to PFS or to model mixtures of excipients and quantified by real-time PCR. The tested excipients evidenced clear adsorption phenomena that justify the hampering effect on DNA extraction from PFS. The use of either 10% talc or 0.5% dyes completely adsorbed DNA, resulting in negative PCR amplifications. For the first time, pharmaceutical excipients were shown to affect DNA extraction explaining the inability of recovering DNA from some PFS samples in previous studies.
•DNA extraction is required for PFS botanical authentication studies.•The adsorption effect of talc, silica and dyes on DNA extraction was evaluated.•Food supplements and model mixtures of excipients were spiked with known amounts of maize DNA.•Maize DNA (internal standard) recoveries were calculated based on real-time PCR results.•Excipients in food supplements can hamper DNA extraction due to adsorption phenomena.
•Universal primers targeting the 16S rRNA gene were designed for crustaceans.•A PCR assay provide a simple and high throughput tool to detect crustaceans.•A probe-specific quantitative real-time PCR ...assay was proposed to detect shrimps.•Sensitivities of 0.1 pg of shrimp DNA and 0.0001% of shrimp in béchamel were reached.•The real-time PCR assay was successfully validated and applied to processed foods.
Allergy to crustaceans is an increasingly important food safety issue. To protect people from experiencing adverse allergic reactions, reliable methodologies are necessary to verify the labelling of processed seafood. In the present work, two new DNA-based approaches targeting the 16S rRNA mitochondrial gene are proposed to detect crustaceans in foods using a qualitative PCR assay specific for crustaceans (shrimps, lobsters and crabs) and a quantitative real-time PCR assay specific for shrimp crustaceans. The real-time PCR system allowed the detection and quantification down to 0.1 pg and 0.0001% (w/w) of shrimp DNA and shrimp in model mixtures, respectively. The method exhibited high performance for quantitative analysis in the range of 0.0001% to 50% as inferred by the calibration curve parameters being effectively validated with blind mixtures. The qualitative PCR assay can provide a simple, fast and high throughput tool for screening the presence of crustaceans in processed foods, while the proposed real-time PCR method proved to be a useful tool for the accurate detection and quantification of shrimp in foods at trace levels.
•HRM analysis targeting COI barcode was developed for hake species differentiation.•Five Merluccius species were discriminated in distinct clusters.•Real-time PCR assay targeting COI gene allowed ...detecting hake DNA down to 0.2–20pg.•The method was successfully applied to analyse processed seafood samples.•Results suggest adulterations based on the substitution of labelled species.
Hake species of Merluccius genus represent an important group of fish commonly sold all over the world. Therefore, they are highly prone to be adulterated, particularly the substitution of M. merluccius by other species with lower market value. The present work intended the development of a highly sensitive methodology for the rapid detection and differentiation of hake species based on mini-barcoding of cytochrome c oxidase subunit I (COI) gene combined with high resolution melting (HRM) analysis. The method allowed the full discrimination of M. merluccius, M. productus, M. hubbsi, M. capensis and M. paradoxus with high levels of confidence. Real-time PCR assay targeting COI mini-barcode provided a high sensitive tool to detect hake species down to 0.2–20pg of DNA with adequate performance parameters. The application of the COI-HRM approach to 45 fish-containing foods showed that two samples did not comply with the declared species, suggesting mislabelling or species substitution. These findings highlight the need of controlling processed fish-containing foods and the feasibility of the proposed tool for their authentication at trace levels.
Penaeidae family includes shrimp species of high commercial value, sharing noticeable morphological similarities, which makes them potential targets of adulteration. Therefore, mechanisms for ...authentication and certification of such crustaceans, frequently included in processed foods, constitute a benefit for the food industry. Litopenaeus vannamei, Penaeus monodon, Fenneropenaeus indicus, Metapenaeus affinis and Melicertus kerathurus are some of the most relevant penaeid shrimps, being their differentiation of high importance. This work intended to develop a new approach for the specific detection and differentiation of those five closely related shrimp species based on high resolution melting (HRM) analysis targeting a cytochrome oxidase subunit I (COI) mini-barcode. The method enabled the differentiation of the five species with high levels of confidence (>99%), being successfully applied to analyse processed seafood samples. F. indicus and L. vannamei were the main identified species in the commercial products. When verifying labelling compliance, four samples suggest adulterations based on the complete or partial substitution of declared species. The proposed method proved to be a potential tool for the rapid and cost-effective differentiation of penaied shrimp species.
•HRM analysis targeting COI gene was developed for penaeid shrimp differentiation.•Five Penaeidae species were discriminated in distinct clusters.•The method was successfully applied to analyse processed seafood samples.•F. indicus and L. vannamei were the main species identified in the samples.•Results suggest adulterations based on the substitution of labelled species.
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