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The increasing number of food frauds, mainly targeting high quality products, is a rising concern among producers and authorities appointed to food controls. Therefore, the development or implementation of methods to reveal frauds is desired. The genetic traceability of traditional or high-quality dairy products (i.e., products of protected designation of origin, PDO) represents a challenging issue due to the technical problems that arise. The aim of the study was to set up a genetic tool for the origin traceability of dairy products. We investigated the use of Short Tandem Repeats (STRs) to assign milk and cheese to the corresponding producer. Two farms were included in the study, and the blood of the cows, bulk milk, and derived cheese were sampled monthly for one year. Twenty STRs were selected and Polymerase Chain Reactions for each locus were carried out. The results showed that bulk milk and derived cheese express an STR profile composed of a subset of STRs of the lactating animals. A bioinformatics tool was used for the exclusion analysis. The study allowed the identification of a panel of 20 markers useful for the traceability of milk and cheeses, and its effectiveness in the traceability of dairy products obtained from small producers was demonstrated. [ABSTRACT FROM AUTHOR]

The fight against fraud in the wine sector requires continuous improvements and validations of new technologies applicable to musts and wines. Starting from published data from the Vitis18kSNP array, a series of new specific single nucleotide polymorphism (SNP) markers have been identified for some important north-western Italian cultivars, such as Barbera, Dolcetto and Arneis (Vitis vinifera L.), used in the production of high-quality wines under Protected Denomination of Origin. A pair of new SNP markers for each grape variety were selected and validated using two real-time PCR techniques: TaqMan® genotyping assays and high-resolution melting analysis (HRM). The TaqMan® assay has proven to be more reliable and repeatable than HRM analysis because despite being an economical and versatile technique for the detection of different types of genomic mutations (SNPs, insertions or deletions), HRM has shown limitations in the presence of poor-quality DNA extracted from musts and wines. TaqMan® assays have successfully identified Barbera, Dolcetto and Arneis in their respective musts and experimental wines, and with good efficiency in commercial wines. Marked differences between genotypes were observed, varietal identification in Dolcetto-based musts/wines was more efficient than that in Arneis-based wines. Therefore, the TaqMan® assay has considerable potential for varietal identification in wines and the procedure described in the present work can be easily adapted to all wines with adequate setup of DNA extraction methods that should be adapted to different wines.

Outreach activities give high school students an opportunity to better understand the techniques and strategies used by researchers. Here is an experience with high school students designed to familiarize them with genetic methodologies. Students have been challenged to discover whether restaurant beef burgers are made with female or male beef. This represents a didactic way to introduce students to genetic traceability methodologies and also to demonstrate the usefulness of these methodologies in relation to food safety and, more importantly, in sustaining consumer confidence. The exercise is planned to be conducted in a one‐day laboratory session. [ABSTRACT FROM AUTHOR]

The increasing number of food frauds, mainly targeting high quality products, is a rising concern among producers and authorities appointed to food controls. Therefore, the development or implementation of methods to reveal frauds is desired. The genetic traceability of traditional or high-quality dairy products (i.e., products of protected designation of origin, PDO) represents a challenging issue due to the technical problems that arise. The aim of the study was to set up a genetic tool for the origin traceability of dairy products. We investigated the use of Short Tandem Repeats (STRs) to assign milk and cheese to the corresponding producer. Two farms were included in the study, and the blood of the cows, bulk milk, and derived cheese were sampled monthly for one year. Twenty STRs were selected and Polymerase Chain Reactions for each locus were carried out. The results showed that bulk milk and derived cheese express an STR profile composed of a subset of STRs of the lactating animals. A bioinformatics tool was used for the exclusion analysis. The study allowed the identification of a panel of 20 markers useful for the traceability of milk and cheeses, and its effectiveness in the traceability of dairy products obtained from small producers was demonstrated.

Abstract The European flat oyster (Ostrea edulis) is a highly appreciated mollusk with an important aquaculture production throughout the 20th century, in addition to playing an important role on coastal ecosystems. Overexploitation of natural beds, habitat degradation, introduction of non‐native species, and epidemic outbreaks have severely affected this important resource, particularly, the protozoan parasite Bonamia ostreae, which is the main concern affecting its production and conservation. In order to identify genomic regions and markers potentially associated with bonamiosis resistance, six oyster beds distributed throughout the European Atlantic coast were sampled. Three of them have been exposed to this parasite since the early 1980s and showed some degree of innate resistance (long‐term affected group, LTA), while the other three were free of B. ostreae at least until sampling date (naïve group, NV). A total of 14,065 SNPs were analyzed, including 37 markers from candidate genes and 14,028 from a medium‐density SNP array. Gene diversity was similar between LTA and NV groups suggesting no genetic erosion due to long‐term exposure to the parasite, and three population clusters were detected using the whole dataset. Tests for divergent selection between NV and LTA groups detected the presence of a very consistent set of 22 markers, located within a putative single genomic region, which suggests the presence of a major quantitative trait locus associated with B. ostreae resistance. Moreover, 324 outlier loci associated with factors other than bonamiosis were identified allowing fully discrimination of all the oyster beds. A practical tool which included the 84 highest discriminative markers for tracing O. edulis populations was developed and tested with empirical data. Results reported herein could assist the production of stocks with improved resistance to bonamiosis and facilitate the management of oyster beds for recovery production and ecosystem services provided by this species.

Wine authentication through grapevine DNA traceability could be affected by wine technological processing treatments. In this study, filtration treatments including depth filter treatment using kieselguhr, perlite and membrane filtration using different types and pore sizes (0.22 and 0.45 μm) were applied on 'Nebbiolo' wine at an experimental scale. We used 'Nebbiolo' because it is an important Italian winegrape variety used to produce high-quality wines. Phenolic composition and color properties of the treated wines were examined using spectrophotometric and HPLC methods, while grapevine DNA traceability was evaluated using PCR based assays. The filtration treatments, as expected, significantly decreased turbidity compared to the unfiltered control, although total phenolics, total flavonoids and total non-flavonoids were not significantly affected. A significant reduction of acetylated anthocyanins by 6.1%–43.3% was observed in filtered wines, which could account for the reduced in total anthocyanins and color intensity of these wines. Grapevine DNA was significantly reduced in filtered wine by 37.2%–99.7%, with the reduction rate depending mainly on the properties of filter material. Polyvinylidene difluoride (PVDF) membranes with a pore size of 0.22 μm showed highest reduction of grapevine DNA in wine, resulting in the failure of TaqMan® single nucleotide polymorphisms (SNPs)-based assays used to detect grape DNA in wines. [Display omitted] • Filtration treatments significantly reduced acetylated anthocyanins in wine. • PVDF 0.22 μm filtration treatment significantly reduced grapevine DNA in wine. • Reduction rate of grapevine DNA was dependent on filter method and material. • SNP-based varietal identification efficiency was influenced by filter pore size. [ABSTRACT FROM AUTHOR]

The fight against fraud in the wine sector requires continuous improvements and validations of new technologies applicable to musts and wines. Starting from published data from the Vitis18kSNP array, a series of new specific single nucleotide polymorphism (SNP) markers have been identified for some important north-western Italian cultivars, such as Barbera, Dolcetto and Arneis ( Vitis vinifera L.), used in the production of high-quality wines under Protected Denomination of Origin. A pair of new SNP markers for each grape variety were selected and validated using two real-time PCR techniques: TaqMan® genotyping assays and high-resolution melting analysis (HRM). The TaqMan® assay has proven to be more reliable and repeatable than HRM analysis because despite being an economical and versatile technique for the detection of different types of genomic mutations (SNPs, insertions or deletions), HRM has shown limitations in the presence of poor-quality DNA extracted from musts and wines. TaqMan® assays have successfully identified Barbera, Dolcetto and Arneis in their respective musts and experimental wines, and with good efficiency in commercial wines. Marked differences between genotypes were observed, varietal identification in Dolcetto-based musts/wines was more efficient than that in Arneis-based wines. Therefore, the TaqMan® assay has considerable potential for varietal identification in wines and the procedure described in the present work can be easily adapted to all wines with adequate setup of DNA extraction methods that should be adapted to different wines., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

The European flat oyster (Ostrea edulis) is a highly appreciated mollusk with an important aquaculture production throughout the 20th century, in addition to playing an important role on coastal ecosystems. Overexploitation of natural beds, habitat degradation, introduction of non‐native species, and epidemic outbreaks have severely affected this important resource, particularly, the protozoan parasite Bonamia ostreae, which is the main concern affecting its production and conservation. In order to identify genomic regions and markers potentially associated with bonamiosis resistance, six oyster beds distributed throughout the European Atlantic coast were sampled. Three of them have been exposed to this parasite since the early 1980s and showed some degree of innate resistance (long‐term affected group, LTA), while the other three were free of B. ostreae at least until sampling date (naïve group, NV). A total of 14,065 SNPs were analyzed, including 37 markers from candidate genes and 14,028 from a medium‐density SNP array. Gene diversity was similar between LTA and NV groups suggesting no genetic erosion due to long‐term exposure to the parasite, and three population clusters were detected using the whole dataset. Tests for divergent selection between NV and LTA groups detected the presence of a very consistent set of 22 markers, located within a putative single genomic region, which suggests the presence of a major quantitative trait locus associated with B. ostreae resistance. Moreover, 324 outlier loci associated with factors other than bonamiosis were identified allowing fully discrimination of all the oyster beds. A practical tool which included the 84 highest discriminative markers for tracing O. edulis populations was developed and tested with empirical data. Results reported herein could assist the production of stocks with improved resistance to bonamiosis and facilitate the management of oyster beds for recovery production and ecosystem services provided by this species. [ABSTRACT FROM AUTHOR]

A colorimetric assay, exploiting the combination of loop-mediated isothermal amplification (LAMP) with DNA barcoding, was developed to address the authentication of some cephalopod species, a relevant group in the context of seafood traceability, due to the intensive processing from the fishing sites to the shelf. The discriminating strategy relies on accurate design of species-specific LAMP primers within the conventional 5’ end of the mitochondrial COI DNA barcode region and allows for the identification of Loligo vulgaris among two closely related and less valuable species. The assay, coupled to rapid genomic DNA extraction, is suitable for large-scale screenings and on-site applications due to its easy procedures, with fast (30 min) and visual readout.

Digital PCR (dPCR) is a breakthrough technology that able to provide sensitive and absolute nucleic acid quantification. It is a third-generation technology in the field of nucleic acid amplification. A unique feature of the technique is that of dividing the sample into numerous separate compartments, in each of which an independent amplification reaction takes place. Several instrumental platforms have been developed for this purpose, and different statistical approaches are available for reading the digital output data. The dPCR assays developed so far in the plant science sector were identified in the literature, and the major applications, advantages, disadvantages, and applicative perspectives of the technique are presented and discussed in this review.

The development of a colorimetric mono-varietal discriminating assay, aimed at improving traceability and quality control checks of durum wheat products, is described. A single nucleotide polymorphism (SNP) was identified as a reliable marker for wheat varietal discrimination, and a rapid test for easy and clear identification of specific wheat varieties was developed. Notably, an approach based on the loop-mediated isothermal amplification reaction (LAMP) as an SNP discrimination tool, in combination with naked-eye visualization of the results, was designed and optimized. Our assay was proven to be effective in the detection of adulterated food products, including both substitution and mixing with different crop varieties.

Pasta, the Italian product par excellence, is made of pure durum wheat. The use of Triticum durum derived semolina is in fact mandatory for Italian pasta, in which Triticum aestivum species is considered a contamination that must not exceed the 3% maximum level. Over the last 50 years, various electrophoretic, chemical, and immuno-chemical methods have been proposed aimed to track the possible presence of common wheat in semolina and pasta. More recently, a new generation of methods, based on DNA (DeoxyriboNucleic Acid) analysis, has been developed to this aim. Species traceability can be now enforced by a new technology, namely digital Polymerase Chain Reaction (dPCR) which quantify the number of target sequence present in a sample, using limiting dilutions, PCR, and Poisson statistics. In our work we have developed a duplex chip digital PCR (cdPCR) assay able to quantify common wheat presence along pasta production chain, from raw materials to final products. The assay was verified on reference samples at known level of common wheat contamination and applied to commercial pastas sampled in the Italian market.

Traceability systems help guarantee that products bought from stores exactly correspond to the description on the label. Traceability maps the journey of the food, from the store, throughout processing, right back to the original livestock. DNA can be used as a natural barcode, as it carries unique genetic information and can be used to identify individual animals. Genetic variability of markers was analysed with the aim of confirmation of the available set of 13 microsatellites. With the addition of five polymorphic markers, the probability of finding two animals sharing the same profile was, on average, four in ten million for the entire dataset. In Slovakia in 2015, 65 126 red deers were recorded. Using only the five extra polymorphic markers, it was possible to obtain a reliable individual genetic traceability system. [ABSTRACT FROM AUTHOR]

Catch and consumption of torpedo scad ( Megalaspis cordyla ) over the western Indian Ocean, but also the western Pacific from Japan to Australia is constantly increasing. Taking into account the degree of exploitation and missing information on the population structure of torpedo scad stocks it is crucial to provide population data. The analysis included individuals obtained in 2012 and 2013 from local markets in Madagascar, Tanzania, Vietnam and Cambodia and after successful DNA extraction fragment of the nuclear rhodopsin gene (RH1) and 9 microsatellite regions (SSRs) were amplified and analysed. Based on the obtained results it was found that there was no 100% overlap between the compared RH1 sequences and those from GenBank. In the case of the studied SSRs, the results allowed the initial characterisation and assessment of the genetic diversity of populations. Moreover, population assignment test distinguished the studied populations into two geographically distant subpopulations. [ABSTRACT FROM AUTHOR]

Genetic traceability has a key role in the product certification, but it is rarely implemented in sheep so far, especially in the fresh meat sector. In this study, the case of the Sambucana sheep is analysed with the aim of developing a genetic system able to certify the origin of its traditional product, the Sambucano lamb, protected by a registered trademark. A set of 14 microsatellite markers was identified as an efficient tool to genetically discriminate the Sambucana sheep from other breeds potentially involved in mislabelling and to allow for an effective allocation test of meat cuts labelled as ‘Guaranteed Sambucano lamb’. The paternity test proved to be an additional means to improve the reliability of the control. The traceability system here described is easy to implement in local minor sheep breeds and is recommended in the framework of meat certification. [ABSTRACT FROM AUTHOR]

DNA barcoding is a molecular technology that allows the identification of any biological species by amplifying, sequencing and querying the information from genic and/or intergenic standardized target regions belonging to the extranuclear genomes. Although these sequences represent a small fraction of the total DNA of a cell, both chloroplast and mitochondrial barcodes chosen for identifying plant and animal species, respectively, have shown sufficient nucleotide diversity to assess the taxonomic identity of the vast majority of organisms used in agriculture. Consequently, cpDNA and mtDNA barcoding protocols are being used more and more in the food industry and food supply chains for food labeling, not only to support food safety but also to uncover food piracy in freshly commercialized and technologically processed products. Since the extranuclear genomes are present in many copies within each cell, this technology is being more easily exploited to recover information even in degraded samples or transformed materials deriving from crop varieties and livestock species. The strong standardization that characterizes protocols used worldwide for DNA barcoding makes this technology particularly suitable for routine analyses required by agencies to safeguard food safety and quality. Here we conduct a critical review of the potentials of DNA barcoding for food labeling along with the main findings in the area of food piracy, with particular reference to agrifood and livestock foodstuffs. [ABSTRACT FROM AUTHOR]

DNA barcoding is a molecular technology that allows the identification of any biological species by amplifying, sequencing and querying the information from genic and/or intergenic standardized target regions belonging to the extranuclear genomes. Although these sequences represent a small fraction of the total DNA of a cell, both chloroplast and mitochondrial barcodes chosen for identifying plant and animal species, respectively, have shown sufficient nucleotide diversity to assess the taxonomic identity of the vast majority of organisms used in agriculture. Consequently, cpDNA and mtDNA barcoding protocols are being used more and more in the food industry and food supply chains for food labeling, not only to support food safety but also to uncover food piracy in freshly commercialized and technologically processed products. Since the extranuclear genomes are present in many copies within each cell, this technology is being more easily exploited to recover information even in degraded samples or transformed materials deriving from crop varieties and livestock species. The strong standardization that characterizes protocols used worldwide for DNA barcoding makes this technology particularly suitable for routine analyses required by agencies to safeguard food safety and quality. Here we conduct a critical review of the potentials of DNA barcoding for food labeling along with the main findings in the area of food piracy, with particular reference to agrifood and livestock foodstuffs.

[Display omitted] • Oenological processing aids and additives played a role in removing DNA from wine. • Treatments with the highest oenological impact caused the highest loss of DNA. • Loss of DNA caused by aging is lower compared to the loss linked to fining agents. • SNP-based assay failed in 'Nebbiolo' wines treated with bentonite and gelatine. 'Nebbiolo' is a well-known grapevine variety used to produce prestigious monovarietal Italian red wines. Genetic traceability is an important tool used to protect the authenticity of high-quality wines. SNP-based assays are an effective method to reach this aim in wines, but several issues have been reported for the authentication of commercial wines. In this study, the impact of the most common commercial additives and processing aids used in winemaking was analysed in 'Nebbiolo' wine using SNP-based traceability. Gelatine and bentonite had the strongest impact on the turbidity, colour and phenolic composition of wines and on residual grapevine DNA. The DNA reduction associated with the use of bentonite and gelatine (>99% compared to the untreated control) caused issues in the SNP-based assay, especially when the DNA concentration was below 0.5 pg/mL of wine. This study contributed to explaining the causes of the reduced varietal identification efficiency in commercial wines. [ABSTRACT FROM AUTHOR]

Simple Summary: Digital PCR is a third-generation technology based on the subdivision of the analytical sample into numerous partitions that are amplified individually. This review presents the major applications of digital PCR (dPCR) technology developed so far in the field of plant science. In greater detail, dPCR assays have been developed to trace genetically modified plant components, pathogenic and non-pathogenic microorganisms, and plant species. Other applications have concerned the study of the aspects of structural and functional genetics. Digital PCR (dPCR) is a breakthrough technology that able to provide sensitive and absolute nucleic acid quantification. It is a third-generation technology in the field of nucleic acid amplification. A unique feature of the technique is that of dividing the sample into numerous separate compartments, in each of which an independent amplification reaction takes place. Several instrumental platforms have been developed for this purpose, and different statistical approaches are available for reading the digital output data. The dPCR assays developed so far in the plant science sector were identified in the literature, and the major applications, advantages, disadvantages, and applicative perspectives of the technique are presented and discussed in this review. [ABSTRACT FROM AUTHOR]

A colorimetric assay, exploiting the combination of loop-mediated isothermal amplification (LAMP) with DNA barcoding, was developed to address the authentication of some cephalopod species, a relevant group in the context of seafood traceability, due to the intensive processing from the fishing sites to the shelf. The discriminating strategy relies on accurate design of species-specific LAMP primers within the conventional 5' end of the mitochondrial COI DNA barcode region and allows for the identification of Loligo vulgaris among two closely related and less valuable species. The assay, coupled to rapid genomic DNA extraction, is suitable for large-scale screenings and on-site applications due to its easy procedures, with fast (30 min) and visual readout. [ABSTRACT FROM AUTHOR]

The development of a colorimetric mono-varietal discriminating assay, aimed at improving traceability and quality control checks of durum wheat products, is described. A single nucleotide polymorphism (SNP) was identified as a reliable marker for wheat varietal discrimination, and a rapid test for easy and clear identification of specific wheat varieties was developed. Notably, an approach based on the loop-mediated isothermal amplification reaction (LAMP) as an SNP discrimination tool, in combination with naked-eye visualization of the results, was designed and optimized. Our assay was proven to be effective in the detection of adulterated food products, including both substitution and mixing with different crop varieties. [ABSTRACT FROM AUTHOR]

Pasta, the Italian product par excellence, is made of pure durum wheat. The use of Triticum durum derived semolina is in fact mandatory for Italian pasta, in which Triticum aestivum species is considered a contamination that must not exceed the 3% maximum level. Over the last 50 years, various electrophoretic, chemical, and immuno-chemical methods have been proposed aimed to track the possible presence of common wheat in semolina and pasta. More recently, a new generation of methods, based on DNA (DeoxyriboNucleic Acid) analysis, has been developed to this aim. Species traceability can be now enforced by a new technology, namely digital Polymerase Chain Reaction (dPCR) which quantify the number of target sequence present in a sample, using limiting dilutions, PCR, and Poisson statistics. In our work we have developed a duplex chip digital PCR (cdPCR) assay able to quantify common wheat presence along pasta production chain, from raw materials to final products. The assay was verified on reference samples at known level of common wheat contamination and applied to commercial pastas sampled in the Italian market. [ABSTRACT FROM AUTHOR]

• 'Nebbiolo'-specific single-nucleotide polymorphisms (SNPs) were identified. • SNP TaqMan® genotyping assays detected 'Nebbiolo' genotype in all wine-making steps. • SNP genotyping assays identified must mixtures at 1% and wine mixtures at 10–20%. • In commercial wines, low-quality DNA limited the efficiency of the SNP assays. • SNPs are promising and user-friendly markers for varietal identification in wine. 'Nebbiolo' (Vitis vinifera L.) is renowned for its use in producing monovarietal high-quality red wines, such Barolo and Barbaresco. The fight against fraud to safeguard high-quality productions requires an effective varietal identification system applicable in musts and wines. 'Nebbiolo'-specific single-nucleotide polymorphisms (SNPs) were identified starting from available databases and 260 genotypes analysed by Vitis18kSNP array. Two SNPs were sufficient to identify 'Nebbiolo' from 1157 genotypes. The SNP TaqMan® genotyping assays developed in this work successfully identified 'Nebbiolo' in all musts and wines collected at different experimental wine-making steps. The high sensitivity of the assays allowed identification of must mixtures at 1% and wine mixtures at 10–20% with non-'Nebbiolo' genotypes. In commercial wines, the amplification efficiency was limited by the low amount of grapevine DNA and the presence of PCR inhibitors. The TaqMan® genotyping assay is a rapid, highly sensitive and specific methodology with remarkable potential for varietal identification in wines. [ABSTRACT FROM AUTHOR]

Catch and consumption of torpedo scad (Megalaspis cordyla) over the western Indian Ocean, but also the western Pacific from Japan to Australia is constantly increasing. Taking into account the degree of exploitation and missing information on the population structure of torpedo scad stocks it is crucial to provide population data. The analysis included individuals obtained in 2012 and 2013 from local markets in Madagascar, Tanzania, Vietnam and Cambodia and after successful DNA extraction fragment of the nuclear rhodopsin gene (RH1) and 9 microsatellite regions (SSRs) were amplified and analysed. Based on the obtained results it was found that there was no 100% overlap between the compared RH1 sequences and those from GenBank. In the case of the studied SSRs, the results allowed the initial characterisation and assessment of the genetic diversity of populations. Moreover, population assignment test distinguished the studied populations into two geographically distant subpopulations., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The genetic varietal authentication of wine was investigated according to DNA isolation procedures reported for enological matrices and also by testing 11 commercial extraction kits and various protocol modifications. Samples were collected at different stages of the winemaking process of renowned Italian wines Brunello di Montalcino, Lambruschi Modenesi, and Trento DOC. Results demonstrated not only that grape DNA loss is produced by the fermentation process but also that clarification and stabilization operations contribute to the reduction of double-stranded DNA content on wine. Despite the presence of inhibitors, downstream PCR genotyping yielded reliable nuclear and chloroplast SSR markers for must samples, whereas no amplification or inconsistent results were obtained at later stages of the vinification. In addition, a TaqMan genotyping assay based on cultivar-specific single-nucleotide polymorphisms (SNPs) was designed, which allowed assessment of grapevine DNA mixtures. Once the wine matrix limitations are overcome, this sensitive tool may be implemented for the relative quantification of cultivars used for blend wines or frauds.