Development of a Simple and Cost-Effective Bead-Milling Method for DNA Extraction from Fish Muscles

In the fish food sector, due to a growing globalization of the market, where intentional and unintentional frauds reach alarming levels, the molecular analysis is increasingly used by both official agencies, to enforce the law on traceability, and private companies, to verify the quality of goods. DNA extraction represents a necessary and critical step for all types of DNA analysis. Among the drawbacks associated with this procedure, there are handling of toxic materials, low DNA yield, and low throughput, due to time-consuming manual procedures. In this work, to overcome some of these problems, we developed an alternative method based on a bead-milling procedure without proteinase K digestion. The new method was then compared with both a salting-out protocol, developed in a previous work, and a commercial kit. Yield, spectrophotometric purity, electrophoretic degradation pattern, and amplificability of the extracted DNA were assessed. In particular, DNA amplificability was evaluated by comparing the band intensity on the gel, after amplification of the 16S rRNA and cytochrome oxidase I genes with a conventional PCR, and the take-off cycles, after amplification of the 16S rRNA gene with a real-time PCR. The results showed that the bead-based method allowed to obtain acceptable amounts of DNA, with good purity and good characteristics of amplificability. Although the salting-out method remains the most effective protocol in terms of pure performances, the bead-milling procedure can be considered a valid alternative, in the light of its lower demand in terms of labor and costs

Fish species identification in canned pet food by BLAST and Forensically Informative Nucleotide Sequencing (FINS) analysis of short fragments of the mitochondrial 16s ribosomal RNA gene (16S rRNA)

Nowadays, pet food claiming high-valued fish among ingredients is largely available on the market. Unfortunately, the modifications induced by processing make species identification by visual inspection difficult and hinder the enforcement of the legislation on traceability. In this work, after aligning 819 sequences of Clupeidae, Engraulidae, Salangidae and Scombridae families, we developed new universal primers for the amplification and sequencing of 2 short fragments (±118 and ±213) of the mitochondrial 16s ribosomal RNA (16S rRNA) gene. Once tested on 130 DNA reference samples, these primers were used in the analysis of highly degraded DNA extracted from 43 canned cat food containing whole minnows (whitebait) (M) and tuna, or bonito or mackerel fillets (F). Three M and 2 F samples were analyzed for each can. A BLAST and a FINS analysis, the latter performed only on the 118 bp fragment, were performed separately on the sequences obtained from M and F samples. All the M samples were identified at the species or genus level by both BLAST and FINS analysis. This allowed to highlight an impressive rate of mislabeling (100%). F samples, for which FINS was less performing in species identification, resulted mislabeled in 40% of the products

A Conventional Multiplex PCR Assay for the Detection of Toxic Gemfish Species (Ruvettus pretiosus and Lepidocybium flavobrunneum): A Simple Method to Combat Health Frauds

The meat of Ruvettus pretiosus and Lepidocybium flavobrunneum (gemfishes) contains high amounts of indigestible wax esters that provoke gastrointestinal disorders. Although some countries have banned the sale of these species, mislabeling cases have been reported in sushi catering. This work developed a simple conventional multiplex PCR, which discriminates the two toxic gemfishes from other potentially replaced species, such as tunas, cod, and sablefish. A common degenerate forward primer and three species-specific reverse primers were designed to amplify cytochrome oxidase subunit I (COI) gene regions of different lengths (479, 403, and 291 bp) of gemfishes, tunas, and sablefish, respectively. A primer pair was designed to amplify a fragment (193 bp) of the cytb gene of cod species. Furthermore, a primer pair targeting the 16S rRNA gene was intended as common positive control (115 bp). The method developed in this study, by producing the expected amplicon for all of the DNA samples tested (reference and commercial), provides a rapid and reliable response in identifying the two toxic species to combat health frauds

Multiple DNA BARCODING for fish species identification in sushi products

The aim of this work was to perform a molecular survey based on DNA barcoding to identify the seafood species used in the preparation of ethnic products (sushi). Twenty-one raw products (each composed of 3 to 8 pieces, for a total of 88 samples) were purchased in ethnic restaurants in the provinces of Pisa (11), Lucca (2), Livorno (3) and Florence (5). The total DNA extracted (1) was evaluated by gel electrophoresis and amplified using universal primers for mitochondrial (COI, 16SrRNA) or nuclear genes (PEPCK) depending on the species (fish, mollusk or crustacean) and the level of DNA degradation. Different primers (2,3,4,5,6,7) for the amplification of a long (~700 bp) or a short (~139-200 bp) fragment were used. Ninety-five PCR products were obtained (for some products two genes were analyzed). Of these, 30 have already been sequenced (Experimental Zooprophylactic Institute of Latium and Tuscany (Rome)). The sequences were elaborated with Clustal W in Bioedit 7.0.9.0, and analyzed by a BLAST analysis on GenBank and by using the Identification System on BOLD. A top match with a sequence similarity of at least 98% was used to designate potential species identification (8). DNA was degraded in almost one third of the samples. This was probably due to rice acidification, to repeated cycles of freezing/thawing or to prolonged storage. The degradation was confirmed by PCR amplification. In fact, we obtained long amplicons in 72.6% of the cases (n=69) and short amplicons for 27.3% of the samples (n=26). The average length of the long sequences was 595 bp for the COI FDB and 490 bp for the PEPCK gene, while the length of the short sequences was ~210bp for the 16S rRNA and 139bp for the COI MDB. All the samples were identified at least at the genus level, with identity values ranging from 99 to 100%. Although for some samples it was impossible to achieve a specific identification, the results were informative enough to verify the information given by the producers. No samples were found mislabeled. Even though the COI gene represents the most exploited target for seafood species identification, issues were found during amplification and comparison with the databases. Thus, in order to increase the PCR output, new universal primers, able to amplify a wide range of taxa, would be desirable. Finally, in case of degraded DNA samples, where the number of diagnostic mutation is limited, a multiple gene analysis is advisable

DNA barcoding reveals chaotic labeling and misrepresentation of cod (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

The uncertainty of seafood labeling in China: A case study on Cod, Salmon and Tuna

Exotic marine fish products are increasingly appreciated in China. In this study, 100 samples of Cod, Salmon and Tuna products were collected from supermarkets in Shanghai, Nanjing and Hangzhou. First the information reported on the label were assessed in the light of the Chinese legislation, paying particular attention to the fish names and the geographical origin. Then, a comparative analysis of the official trade denominations adopted by five European countries (Italy, France, Germany, Spain and United Kingdom) for Cod, Salmon and Tuna was performed. Finally, the Chinese names of the species considered in the EU list were verified consulting the available international lists. Overall, 95% of the samples employed just generic names. In particular, 98% of Salmon and 100% of Tuna products were generically labeled while the labeling of Cod products was more diversified, even though 80% reported misleading or fake denominations. The results of this work highlighted the lack of a mandatory legislation on seafood traceability and of an official naming system. In particular, this study propose the introduction of a detailed Chinese naming system based on the Chinese Latin Dictionary for Seafood Names, following the EU approach. In fact, inaccurate labeling can have both economic and health implications for consumers as well as it may distort the true abundance of fish stocks. These drawbacks can be particularly serious considering the pivotal role of China in the global fishery industry

Pentaplex PCR as screening assay for jellyfish species identification in food product

Salted jellyfish, a traditional food in Asian Countries, is nowadays spreading on the Western markets. In this work, we developed a Pentaplex PCR for the identification of five edible species (Nemopilema nomurai, Rhopilema esculentum, Rhizostoma pulmo, Pelagia noctiluca, and Cotylorhiza tuberculata), which cannot be identified by a mere visual inspection in jellyfish products sold as food. A common degenerated forward primer and five specie-specific reverse primers were designed to amplify COI gene regions of different lengths. Another primer pair targeted the 28SrRNA gene and was intended as common positive reaction control. Considering the high level of degradation in the DNA extracted from acidified and salted products, the maximum length of the amplicons was set at 200 bp. The PCR was developed using 66 reference DNA samples. It gave successful amplifications in 85.4% of 48 ready to eat products (REs) and in 60% of 30 classical salted products (CPs) collected on the market

Evolution of the Anisakis risk management in the European and Italian context

Due to the social and legislative implications, the presence of Anisakis spp. larvae in fishery products has become a concern for both the consumers and the official Control Authorities. The issuance of a large number of provisions, aimed at better managing fish products intended to be consumed raw or almost raw and the associated risks, resulted in a very complicate legal framework. In this work, we analyzed the evolution of the normative through an overview on the local and international legislations, focusing on issues that are of practical interest for Food Business Operators (FBOs) in the fishery chain. In addition, we performed a survey across the Department of Prevention of the Italian Local Health Authorities (LHA) and the main fish markets in Italy to collect the operating procedures and the monitoring plans. Overall, we found many differences, due to the absence of a national reference standard for the management of the Anisakis risk. From this examination, it turns clear that only a participation of all the involved institutions, a strategy of synergistic interventions, as well as a correct training of FBOs, can result in an effective risk management and a proper risk communication, which should overcome states of confusion and unnecessary negative impacts on the economy

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

Assessment of a Sampling Plan Based on Visual Inspection for the Detection of Anisakid Larvae in Fresh Anchovies (Engraulis encrasicolus). A First Step Towards Official Validation?

The presence of anisakid larvae in fish is a public health issue, and effective risk management procedures are needed to avoid that heavily infected products reach the market. Currently, an official sampling plan for fresh fish defining sample size, inspection methods, and criteria to accept or reject the merchandise is lacking at the European and Italian level. In this study, we compared the visual inspection proposed by the sampling plan of the Lombardy Region (Italy) to the UV press method and to an optimized digestion procedure with the aim to assess its ability in detecting visible parasites. Thirty-one batches of Engraulis encrasicolus, each composed of ∼30 specimens, were collected and subsequently analyzed with the three techniques. The mean abundance (MA) was calculated after each procedure and compared on the basis of a threshold value. The results showed that the visual inspection performed similarly to the digestion method, with a sensitivity of 93 %, a specificity of 100 %, and an accuracy of 97 %. Overall, the comparison showed that, in the proposed sampling plan, the visual inspection is effective in rejecting unmarketable anchovies and in preventing the commercialization of unsafe products. This method is simple, less demanding than digestion in terms of time and equipment, and thus suitable as a standardized procedure to be routinely applied by food business operators. The hazard characterization, performed by sequencing the mtDNA cox2 gene, has identified the visible larvae as Anisakis pegreffii in 98 % of the cases, highlighting the zoonotic potential of the parasites found and the need for preventive measure