DNA and Mini-DNA barcoding for the identification of Porgies species (family Sparidae) of commercial interest on the international market

The morphological similarity among Sparidae species, which are characterized by a different market price, represents a serious problem for their trade and for stock management, since it encourages frauds for substitution. The most accredited morphological method for their identification is based on the dental-plate, but this approach is not simple and cannot be used for prepared products. When molecular methods are used the DNA degradation induced by cooking is the main drawback. In this work, we collected 314 reference tissues belonging to 75 Sparidae species and we produced a dataset of full (FDB) and mini-barcode (MDB) reference sequences starting from DNA extracted from fresh and ethanol-preserved tissues using universal primes. Moreover, some fresh samples were cooked. The FDB was successfully amplified in 91% (fresh), 50% (cooked) and 81% (ethanol-preserved) samples, while the amplification rates of the MDB were considerably higher in case of cooked (100%) and ethanol-preserved (94%) samples. The same primers were used for the amplification of the DNA obtained from 58 market samples (MS). All the DNA barcodes were compared with BOLD and GenBank using IDs and BLAST analysis. FDB was able to provide unambiguous species-level identifications for 53 (78%) and 44 (64.7%) reference samples analyzed on BOLD and GenBank, respectively. The Mini-DNA barcode (MDB) showed a lower discriminating power with 32 (45.7%) and 29 (41.4%) sequences unambiguously matched to a species on BOLD and GenBank. However, the MDB allowed to identify all the reference sequences as belonging to the Sparidae family. FDB and MDB showed a similar performance in analyzing the MS, allowing to highlight 21 (38%) mislabeled MS. Our study, while confirming the FDB as a reliable tool for fish authentication, proposes the MDB as a promising tool to recover molecular information in case of cooked products.     

Novel nuclear barcode regions for the identification of flatfish species

The development of an efficient seafood traceability framework is crucial for the management of sustainable fisheries and the monitoring of potential substitution fraud across the food chain. Recent studies have shown the potential of DNA barcoding methods in this framework, with most of the efforts focusing on using mitochondrial targets such as the cytochrome oxidase 1 and cytochrome b genes. In this article, we show the identification of novel targets in the nuclear genome, and their associated primers, to be used for the efficient identification of flatfishes of the Pleuronectidae family. In addition, different in silico methods are described to generate a dataset of barcode reference sequences from the ever-growing wealth of publicly available sequence information, replacing, where possible, labour-intensive laboratory work. The short amplicon lengths render the analysis of these new barcode target regions ideally suited to next-generation sequencing techniques, allowing characterisation of multiple fish species in mixed and processed samples. Their location in the nucleus also improves currently used methods by allowing the identification of hybrid individuals.     

DNA barcoding reveals commercial and health issues in ethnic seafood sold on the Italian market

The number of seafood species sold on Western markets is constantly growing and many unconventional species are sold in ethnic food outlets. In this work, 68 ethnic seafood products variously processed were collected from the Italian market and a molecular analysis was performed by sequencing a full cytochrome c oxidase (COI) DNA barcode (FDB, ∼655 bp) or a mini COI DNA barcode (MDB, ∼139 bp) using universal primers. Barcodes were then compared with sequences available in BOLD and GenBank. In addition, the label information was assessed according to the European legislation. By using the IDs analysis on BOLD a maximum species identity ≥98% was retrieved for 84% of the sequences. Of these, 67% were unambiguously identified at species level (51.3% of the FDB and 74% of the MDB). Using NCBI BLAST, 74% of the sequences scored a maximum species identity ≥98%, of which 73% were identified at species level (52% of the FDB and 61% of the MDB). Both databases performed better in mollusk identification. Overall, 45 products (66%) were not correctly labeled according to the European requirements. Finally, the comparison between the molecular and the label analysis highlighted that 48.5% of the products presented discrepancies between labeling and molecular identification. In particular, health implications were highlighted for 2 samples labeled as squid but identified as Lagocephalus spp., a poisonous puffer fish species banned from the EU market. The present results confirm DNA barcoding as a reliable tool for protecting consumers' health and economic interests.     

Identification of meat and poultry species in food products using DNA barcoding

DNA barcoding is a promising method for the sequencing-based identification of meat and poultry species in food products. However, DNA degradation during processing may limit recovery of the full-length DNA barcode from these foods. The objective of this study was to investigate the ability of DNA barcoding to identify species in meat and poultry products and to compare the results of full-length barcoding (658 bp) and mini-barcoding (127 bp). Sixty meat and poultry products were collected for this study, including deli meats, ground meats, dried meats, and canned meats. Each sample underwent full and mini-barcoding of the cytochrome c oxidase subunit I (COI) gene. The resulting sequences were queried against the Barcode of Life Database (BOLD) and GenBank for species identification. Overall, full-barcoding showed a higher sequencing success rate (68.3%) as compared to mini-barcoding (38.3%). Mini-barcoding out-performed full barcoding for the identification of canned products (23.8% vs. 19.0% success), as well as for turkey and duck products; however, the primer set performed poorly when tested against chicken, beef, and bison/buffalo. Overall, full barcoding was found to be a robust method for the detection of species in meat and poultry products, with the exception of canned products. Mini-barcoding shows high potential to be used for species identification in processed products; however, an improved primer set is needed for this application.     

Identification of species in ground meat products sold on the U.S. commercial market using DNA-based methods

The objective of this study was to test a variety of ground meat products sold on the U.S. commercial market for the presence of potential mislabeling. Forty-eight ground meat samples were purchased from online and retail sources, including both supermarkets and specialty meat retailers. DNA was extracted from each sample in duplicate and tested using DNA barcoding of the cytochrome c oxidase I (COI) gene. The resulting sequences were identified at the species level using the Barcode of Life Database (BOLD). Any samples that failed DNA barcoding went through repeat extraction and sequencing, and due to the possibility of a species mixture, they were tested with real-time polymerase chain reaction (PCR) targeting beef, chicken, lamb, turkey, pork and horse. Of the 48 samples analyzed in this study, 38 were labeled correctly and 10 were found to be mislabeled. Nine of the mislabeled samples were found to contain additional meat species based on real-time PCR, and one sample was mislabeled in its entirety. Interestingly, meat samples ordered from online specialty meat distributors had a higher rate of being mislabeled (35%) compared to samples purchased from a local butcher (18%) and samples purchased at local supermarkets (5.8%). Horsemeat, which is illegal to sell on the U.S. commercial market, was detected in two of the samples acquired from online specialty meat distributors. Overall, the mislabeling detected in this study appears to be due to either intentional mixing of lower-cost meat species into higher cost products or unintentional mixing of meat species due to cross-contamination during processing.     

Authentication and traceability of fish maw products from the market using DNA sequencing

Fish maws (dried swimbladders of fishes) are regarded as traditional luxurious delicacies, medicine and tonics, which have been recommended and consumed in Asia over many centuries. At the commercial level, they are ranked as different values based on species. However, fish maw species and the trade are still unknown and undocumented. The processing treatments make them difficult for species identification based on morphological characterization. In the present study, the genetic identification of the main commercialized species of fish maws has been carried out, based on the amplification of a fragment of mitochondrial 16S rRNA gene and subsequent BLAST analysis. The applicability of all kinds of processed products was verified, including dried, water soaked, salt fried and salt fried plus water soaked forms. The result indicated this method was applicable to all of them, showed that 53.3% of the products were incorrectly labeled and 58.3% of “croaker” products were substituted with catfish or perch species. Moreover, results indicated that besides traditional trade in Asia, the fish maw trade has been globally expanded to meet the growing demand of market. Therefore, this method can be useful in normative control of processed products, particularly in the authenticity of imported species, verifying the correct traceability in commercial trade, the correct labeling, and also for fisheries control of endangered species to conserve stocks biodiversity.     

DNA barcoding reveals substitution of Sablefish (Anoplopoma fimbria) with Patagonian and Antarctic Toothfish (Dissostichus eleginoides and Dissostichus mawsoni) in online market in China: How mislabeling opens door to IUU fishing

China’s rapid economic development has determined profound changes in seafood consumption patterns, and nowadays besides the traditional luxury seafood, high-quality marine fish are consumed. Among these is Anoplopoma fimbria (Sablefish), a highly priced species on the Chinese market. A recent molecular survey on products sold online in China found that all the analyzed products sold as Yin Xue, used to indicate A. fimbria, were instead Dissostichus spp., a genus of fish extremely vulnerable to overfishing (Xiong et al., 2016). Considering this and the lack of a standardized naming system for seafood species in China, an initial search was conducted to identify all the possible Chinese names indicating A. fimbria. The aim of the present study was to assess the challenges of the online market with regards to frauds for fish species substitution. DNA barcoding was employed to verify the identity of 42 products sold on e-commerce platforms as Sablefish. Moreover, the information reported on the webpage and on the label was analyzed according to the Chinese regulation in force. All the PCR products gave readable sequences. By using the IDs analysis on BOLD and the BLAST analysis on GenBank all the samples were unambiguously identified at the species level. Of the 42 products sold as Sablefish, only 6 (14.3%) were molecularly identified as this species, while 32 (76.2%) were identified as Dissostichus eleginoides (Patagonian Toothfish) and 4 (9.5%) as Dissostichus mawsoni (Antarctic Toothfish), highlighting an alarming overall misrepresentation rate of 85.7% and implications for the management of these species’ fisheries. The combined analysis of all the information of the webpages and the labels allowed us to hypothesize unintentional and intentional mislabeling. Our findings suggest the possible existence of a trade pattern enabling IUU fishing operators to launder illegal catches of Toothfish through mislabeling.     

Rapid and reliable species identification of scallops by MALDI-TOF mass spectrometry

Correct identification of scallop species is of crucial importance to the seafood industry to assess marketability of the product, as well as to prevent unintentional mislabeling and to comply with current legislative guidelines. However, various scallop species are virtually indistinguishable and to date there is no fast, reproducible, and inexpensive technique for species identification of scallops. A new promising tool in the identification of various organisms is the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). In this study, we aimed to 1) evaluate the possibility of MALDI-TOF MS as a tool for rapid and reliable species identification of scallops and 2) assess the occurrence of mislabeled scallops sold in Switzerland. We therefore used 16 scallop reference samples, as well as scallops sampled at eight Swiss retail facilities and identified the scallop species both by 16S rRNA PCR and MALDI-TOF MS. For all tested scallop species, MALDI-TOF MS yielded reproducible and unique mass spectra profiles covering a wide molecular mass range of 3000–30,000 Da. Species-specific biomarker protein mass patterns were determined. Species identification of reference scallop samples using MALDI-TOF MS was in accordance with species identification using the established 16S rRNA PCR tool. Identification of the scallop samples purchased in Switzerland also yielded identical results for MALDI-TOF MS and the 16S RNA PCR approach. We were able to show that several of the scallop samples were labeled incorrectly. All mislabeled scallops belonged to the species Placopecten magellanicus, but were marketed as “Jacobsmuschel”, a commercial name reserved for Pecten spp. In this study, we show that MALDI-TOF MS allows for rapid, reliable, and highly accurate species identification of scallops.     

Detection of mislabelled seafood products in Malaysia by DNA barcoding: Improving transparency in food market

Seafood mislabelling is a global issue following the increasing worldwide seafood trade particularly of processed seafood products, as well as a general lack of regulations and tight enforcement in some countries. This study is a pioneering seafood forensics survey conducted in Malaysia. A total of 62 seafood samples, either raw, frozen or variously processed, were collected from commercial sources. Molecular analyses were performed by sequencing Full DNA Barcoding (FDB) with target region of ∼700 bp or Mini Barcoding (MDB) with smaller target region of only ∼150 bp. The DNA barcode sequences obtained were compared with those available on BOLD and GenBank databases. The DNA targets were successfully amplified and sequenced from 81% of seafood samples. Among these samples, 16% were found to have been mislabelled at source. This study supports the view that DNA barcoding can be a powerful tool in seafood forensics.     

A novel minisequencing test for species identification of salted and dried products derived from species belonging to Gadiformes

The aim of the present study is to develop an assay for the specific identification of Gadus morhua, Gadus macrocephalus, Gadus ogac, Molva molva and Brosme brosme targeting sequences of the cytochrome b (cyt b) gene of mitochondrial DNA. The primers used in the preliminary PCR were designed in well conserved regions upstream and downstream of the diagnosis sites. They successfully amplified a conserved 188 bp region from the cyt b gene of all the species taken into consideration. The sites of diagnosis have been interrogated using a minisequencing reaction and capillary electrophoresis. All the results of the test were confirmed by fragment sequencing.     

A multiplex PCR assay for species identification of raw and cooked bonito

Attempts are made to establish one-step multiplex PCR assay for distinguishing five species of raw and cooked bonito including Euthynnus pelamis, Euthynnus affinis, Auxis rochei, Auxis thazard, and Sarda orientalis. After constructing the 1141 bp complete mitochondrial cytochrome b genes of five bonito species and other five contrastive Scombridae species, five sets of species-specific primer were designed to amplify different cytochrome b gene fragments in each species individually. The amplified lengths of fragments were respectively 143 bp for A. rochei, 236 bp for E. pelamis, 318 bp for A. thazard, 398 bp for E. affinis and 506 bp for S. orientalis, which could be obviously differentiated from each other on DNA electrophoresis. The five sets of species-specific primer were mixed and applied to simultaneously detect bonito species. All species from 12 commercial raw fish and five species out of eight cooked bonito fillets were successfully identified by the multiplex PCR assay. Experiments carried out demonstrate that the multiplex PCR assay was useful for identifying species of non-overheating fish product.     

Molecular identification of honey entomological origin based on bee mitochondrial 16S rRNA and COI gene sequences

The increasing recognition and production of stingless bee honey has raised the needs to develop rapid, sensitive and accurate methodologies for honey identification through bees' species from the DNA of bees present in honey. Genetic identification of raw honeys produced by different bee species from Malaysia was performed based on mitochondrial DNA sequences and phylogenetic analysis by means of forensically informative nucleotide sequencing (FINS) technique. The 300 bp of mitochondrial large subunit ribosomal RNA (16S rRNA) gene region and mitochondrial cytochrome c oxidase subunit I (COI) gene region were the gene markers used to classify and identify honey produced by Apis honey bees and Trigona stingless bees. The genetic identities of honey origin from Apis dorsata, Apis mellifera, Apis cerana, and Heterotrigona itama were accurately identified. Kelulut honey sourced from the stingless bee was distinguishable from other Apis type of honeys in phylogenetic analyses. This FINS technique is a specific and reliable method for identification of honey origin via its bee source that can be used to overcome the issue of false declaration of honey origin and mislabelling.     

Molecular identification of grouper species using PCR-RFLP technique

A PCR-RFLP method was used to identify five species of raw and processed groupers belonging to the genus, Epinephelus viz. E. areolatus, E. bleekeri, E. faveatus, E. longispinis and E. undulosus. DNA extracted from all the species using phenol-chloroform method amplified the mt 16S rRNA gene, unique for the genus, Epinephelus, using GenF and EpiR primers at 300 bp. This fragment did not amplify the DNA of even closely related grouper genus, Cephalopholis, making the method more specific the grouper genus, Epinephelus. The specificity was further ascertained by performing the internal control that targeted 18S rRNA, which was eukaryotic specific. Digestion of PCR products with three selected restriction enzymes viz. SduI, BciVI, and Sau3AI followed by PAGE yielded species specific patterns that enabled identification of three grouper species viz. E. bleekeri, E. faveatus and E. longispinis, while the other two species viz. E. areolatus and E. undulosus gave similar pattern in the gel, although minor variations in the molecular sizes of digested products were noticed. Chilled, frozen and cooked groupers also gave similar PCR-RFLP pattern to that of raw groupers making this method suitable even for processed products.     

Species identification of commercial jerky products in food and feed using direct pentaplex PCR assay

The direct pentaplex PCR assay was developed for simultaneous identification using species-specific primer sets and a universal eukaryotic primer set in processed jerky products without DNA extraction. The specific primer sets of target meat species amplified the expected 83-, 133-, 166-, and 204-bp PCR products for pork, chicken, beef, and duck, respectively, and obtained no cross-reactivity against a total of sixteen animal species. A universal eukaryotic primer set amplified a 99-bp conserved fragment in all meat species. To evaluate the sensitivity of this assay, the different percentages of jerky samples were prepared with the meat species having the possibility to be mixed. Adulterated beef jerky samples contaminated with 0.1, 0.5, 1, 5, 10, and 50% pork and adulterated duck jerky samples contaminated with 0.1, 0.5, 1, 5, 10, and 50% chicken were prepared in a laboratory. The detection level of direct pentaplex PCR was below 0.1% pork in adulterated beef jerky and 0.1% chicken in adulterated duck jerky. The optimized assay was also applied to the analysis of commercial food and feed jerky products. The meat species in commercial jerky products were successfully identified without DNA extraction.     

DNA barcoding reveals chaotic labeling and misrepresentation of cod (鳕, Xue) products sold on the Chinese market

The increasing rate of seafood frauds, especially in the case of highly priced species, highlights the need of verifying the identity of fish products. This paper describes the application of DNA barcoding to the identification of 52 products commercialized with the Chinese term 鳕 (Xue, Cod) in supermarkets (Nanjing and Shanghai) and in the online market. Considering the lack of harmonization around the definition of Cod, the mislabeling rate was assessed according to three increasingly stringent definitions: Cod meaning Gadiformes species; Cod meaning Gadus spp.; Cod not meaning any specific species, since a qualifier (“Atlantic”, “Pacific” or “Greenland”) should be added in order to refer to Gadus morhua, Gadus macrocephalus or Gadus ogac, respectively.Results highlighted a very high mislabeling rate, which exceeded 60% even with the less stringent definition. Interestingly, only 42.3% of the samples were Gadiformes, while the others were Perciformes, Pleuronectiformes or toxic Tetraodontiformes species. Economic, ecological and health issues arising from the misuse of the term Cod are discussed in the light of the leading role of China in the seafood worldwide industry and of the increased national consumption of marine species.     

Species identification of dried scallop adductor muscle (yao zhu) products sold on the market

Dried scallop adductor muscle, known as yao zhu, has been ranked in the list of the eight sea treasures in Chinese cuisine, it has been commonly recommended and consumed in China over many centuries due to its delicious taste and abundant nutrition. However, the true species composition of the dried scallop adductor muscles in the trade is unclear. Indeed, it is difficult to identify the scallop species just based on their morphological characters of processed adductor muscles. In this study, 60 yao zhu products commercialized in Chinese market were analyzed in order to authenticate species. Direct sequencing and BLAST method based on a fragment of the 16S rRNA gene were carried out and results showed that 34 products (56.7%) were scallop species Argopecten irradians, and other 26 products (43.3%) were the scallop species Mizuhopecten yessoensis, indicated the yao zhu market were dominated by the both species, which were the introduced aquaculture scallop species. Moreover, a simple and fast PCR method proved to be capable of unequivocal identification between the two species. Therefore, this work facilitates the identification of yao zhu products in the market, support of food control and protection of consumers’ rights.     

Identification of moulds from the Taleggio cheese environment by the use of DNA barcodes

The aim of this work was to characterize the mould populations of the Taleggio PDO cheese surface by a combination of culture dependent and independent techniques. DNA samples were extracted: (i) from isolates derived from brine and cheeses at various ripening midpoints, (ii) directly from the surface of commercial cheeses made in different dairies. DNA was examined by PCR at various loci (DNA barcodes). Amplified regions were both ribosomal (ITS1, ITS2 and the D1/D2 region of the 26S rRNA gene) and non-ribosomal (portions of the β-tubulin and EF1α genes). The mould community appeared to be dominated by a species that alone represents 87% of the sequences obtained using the β-tubulin barcode, and is 99% homologous to Penicillium commune. It has been obtained, always identical, from brine, from cheeses at the various ripening midpoints, and from all the commercial cheeses. It presumably represents a new variant of P. commune typical of Taleggio. Three isolates were identified as belonging to three different species of the genus Cladosporium: Cladosporiumoxysporum and two potential new species, detected on the basis of their β-tubulin sequences. Finally, 2 isolates were identified as Aureobasidium pullulans and Eutypella scoparia. This is the first characterization of the mould species for this important Italian PDO cheese production, and is one of the first reports of molecular characterization by means of DNA barcodes on mould DNA directly extracted from cheese without culturing.     

A new set of 16S rRNA universal primers for identification of animal species

In this study, bioinformatics were used to specifically design universal primers within 16S rRNA gene according to the following criteria: the priming sites needed to be sufficiently conserved to permit a reliable amplification (pooled samples) and the genetic marker needed to (a) be sufficiently variable to discriminate among most species and sufficiently conserved within than between species, (b) be short enough to allow also accurate amplification from processed samples (food) and non-invasive approaches (fur, feathers, faeces, etc.) (c) convey sufficient information to assign samples to species and (d) be amplified under variable lab conditions and protocols. Furthermore, short sequences allow the accurate massive inter- and intra-species identification of point mutations by the SSCP technique. The size of the amplified segment ranged from 222 to 252 bp. Amplification and identification success were 100% with all kinds of tissue tested in both raw and processed samples in a wind range of species, mammals (n = 27), fishes (n = 32) birds (n = 19), coleoptera (n = 23), reptiles (n = 5), crustaceans (n = 5) and cephalopods (n = 2), including almost all European mammal and avian game species. In addition, no intra-specific polymorphism was detected. Finally, gene fragments, homologous to those amplified by the primers used herein and retrieved from the GenBank for three animal sets [mammals (n = 248), birds (n = 231) and fishes (n = 644)] showed a particular precise percentage of correct identifications. Therefore, this short segment of the 16S rRNA mitochondrial gene could be a good candidate for a rapid, accurate, low-cost and easy-to-apply and interpret method to identify mammal and avian game species by PCR amplification and sequencing that can be easily incorporated in integrated conservation and forensic programmes.     

Current methods for seafood authenticity testing in Europe: Is there a need for harmonisation?

Mislabelling of food products has recently received a great deal of public scrutiny, but it remains unclear exactly what methods are being utilised in laboratories testing the authenticity of foods. In order to gain insight into the specific area of the analysis of seafood, a questionnaire focussing on the taxonomic groups typically analysed and the techniques utilised was sent to over one hundred accredited laboratories across the UK, Ireland, Spain, Portugal, France and Germany. Forty-five responded positively, demonstrating significant differences in both the species analysed and methods utilised among the countries included in the survey. Indeed, a diversity of methods was employed across laboratories and efforts to harmonise and/or standardise testing were evident only at national scale. This contrasts with the EU wide scale of regulation on seafood labelling, and may lead to inconsistencies in the results produced in countries.     

Labelling compliance assessment and molecular authentication of Grilled Fish Products Sold on Chinese e-commerce: Traceability issues related to the use of umbrella trade names

The present study aimed to verify the labelling compliance, the accuracy of the trade names, and to molecularly identify 74 pre-packaged grilled fish products purchased online on Taobao, the largest e-commerce platform in China. The labelling compliance was assessed in the light of the Chinese standard GB7718-2011 for pre-packaged foods. Products identification was performed by DNA barcoding targeting two mitochondrial genes (COI and cytb) while, the accuracy of the trade name, was assessed by analysing the results obtained using the ‘common name search tool’ available on the FAO FishBase portal. The trade name reported on the label was considered: 1) species-specific when only a species resulted from the search; 2) genus specific when more than one species belonging to the same genus resulted from the search; 3) umbrella term when more than one species belonging to different genera and/or families and orders resulted from the search. These results were compared to the molecular results, and the products were declared not-matching if the molecular results did not match the scientific names obtained by querying the trade name to FishBase portal. All products were fully compliant to the national labelling standard. Species identification was achieved for 97.3% of the tissue samples tested belonging to 72 products by using the two targets (COI and cytb). All products were sold using 12 different trade names consisting of two generic not informative terms (marine fish, fish), seven umbrella terms (eel, anglerfish, sardine, Navodon, snapper, leatherjacket, lionfish) which framed the products in a taxonomic family/order or several taxonomic orders, two genus specific terms (Bombay duck, Yellow croaker) and one species specific term (Tanaka's snailfish). A final mismatching rate of 48.6% was highlighted. The lack of accuracy in the trade names utilization highlighted in this study may favor substitution phenomena with economic and potential environmental impact.