Validation of a tRNA-Glu-cytochrome b key for the molecular identification of 12 hake species (Merluccius spp.) and Atlantic Cod (Gadus morhua) using PCR-RFLPs, FINS, and BLAST

The goal of this study was to develop a diagnostic key for hake meat to solve the limitations of previous identification methodologies, mainly related to the high degradation of the DNA recovered from processed foods. We describe the development of two molecular tools based on polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphisms of the cytochrome b gene, respectively, to identify DNA from 12 hake species in commercial products. The first assay is an exclusion test consisting of the PCR amplification of a 122 bp fragment using nested primers interspecifically conserved in Merluccius spp. and in Gadus morhua. This 122 bp amplicon, being the shortest one so far designed for hake DNA, is a useful traceability tool for highly degraded samples because its sequence contains enough interspecific diagnostic variation to identify 10 hake species and cod and has been successfully amplified from most commercial products so far tested. The second identification key follows a positive outcome of the exclusion test and consists of the PCR amplification of a 464-465 bp fragment and its digestion with three restriction enzymes whose targets map at interspecifically nonconserved sites of the cytochrome b. The key presented here has passed through a rigorous methodological calibration including its testing for genus specificity, its validation on a large number of authenticated sample types from each species range, and its implementation with a maximum likelihood method for the assignment of unknown samples. Together, these two procedures constitute the most complete molecular key so far developed for Merluccius spp., which is optimal for routine identification of hakes in large commercial samples at a reasonable cost-time ratio.     

Identification of shark species in seafood products by forensically informative nucleotide sequencing (FINS)

The identification of commercial shark species is a relevant issue to ensure the correct labeling of seafood products, to maintain consumer confidence in seafood, and to enhance the knowledge of the species and volumes that are at present being captured, thus improving the management of shark fisheries. The polymerase chain reaction was employed to obtain a 423 bp amplicon from the mitochondrial cytochrome b gene. The sequences from this fragment, belonging to 63 authentic individuals of 23 species, were analyzed using a genetic distance method. Nine different samples of commercial fresh, frozen, and convenience food were obtained in local and international markets to validate the methodology. These samples were analyzed, and sequences were employed for species identification, showing that forensically informative nucleotide sequencing (FINS) is a suitable technique for identification of processed seafood containing shark as an ingredient. The results also showed that incorrect labeling practices may occur regarding shark products, probably because of incorrect labeling at the production point.     

ITS1-rDNA-based methodology to identify world-wide hake species of the Genus Merluccius

Species-specific DNA-based tags are valuable tools for the management of both fisheries and commercial fish products. In this study, we have developed a two-step molecular tool to detect the presence of hake DNA (Merluccius spp.) and to identify the exact hake species present in an blind sample. The first test involves PCR amplification of an ITS1-rDNA fragment of 193 bp using nested primers that are interspecifically conserved in Merluccius spp. and Atlantic cod, Gadus morhua. The second test consists of the PCR amplification of a 602-659 bp DNA fragment spanning part of the ribosomal cluster 18S-ITS1-5.8S and digesting it with four restriction enzymes whose targets map at interspecifically nonconserved sites of the ITS1. Alternatively, the identification of hake species can be achieved by FINS or BLAST, using the nucleotide sequence of either the whole ITS1 sequence or its nested fragment of 193 bp. Because of their high reproducibility and ease of execution, these procedures allow for routine analysis and constitute high reliable tools for the rapid identification of 12 species of hake.     

Direct sequencing method for species identification of canned sardine and sardine-type products

A direct sequencing method based on a 103 bp diagnostic sequence derived from a species-specific mitochondrial DNA cytochrome b sequence of 150 bp obtained by Polymerase Chain Reaction was tested for the identification of 47 commercial canned sardine and sardine-type products from various countries. Multiple alignment of 14 analyzed reference samples belonging to Clupeomorpha species was performed versus the canned samples. Low intraspecific variability was observed for canned sardine (相似文献   

Identification of flatfish (Pleuronectiforme) species using DNA-based techniques

Identification of flatfish species using a DNA-based methodology was studied. The polymerase chain reaction was employed to obtain a 464 bp amplicon from mitochondrial cytochrome b gene. The sequences from this fragment belonging to 24 species were analyzed using a genetic distance method, and polymorphic sites were determined. The fragment was found to be highly polymorphic (231 sites), and this permitted the differentiation of most of the species. Phylogenetic tree construction was employed to allow the identification of flatfish species. As a result, each species was grouped in a well-differentiated clade, except for two pairs: Limanda ferruginea and L. limanda, and Solea impar and S. lascaris, which could not be differentiated. On the basis of the sequences obtained, restriction enzymes were selected to provide specific restriction profiles, which allow the differentiation of 21 species of flatfish in a faster and less expensive manner than sequencing. This polymerase chain reaction-restriction fragment length polymorphism methodology (PCR-RFLP) was tested using commercial samples.     

Detection of mislabeling in hake seafood employing mtSNPs-based methodology with identification of eleven hake species of the genus Merluccius

Species-specific DNA-based tags are valuable tools for the management of both fisheries and commercial fish products. Eleven hake species of the genus Merluccius have been identified employing mtSNPs-based methodology. The method is highly reproducible, fast, and technically easy. It is a reliable tool, allowing for routine analysis of commercial seafood. It can be applied by nonexperts in genetics because both laboratory handling and interpretation of results are easy and direct. The convenience of routine surveys in fish markets has been clearly established with a survey of commercial hake batches imported in south Europe. A total of 40 commercial processed hake were analyzed in this study. More than 20% of mislabeling has been detected.     

Genetic identification of horse mackerel and related species in seafood products by means of forensically informative nucleotide sequencing methodology

In the present study, a methodology based on the amplification of a fragment of mitochondrial cytochrome b and subsequent phylogenetic analysis (FINS: forensically informative nucleotide sequencing) to genetically identify horse mackerels have been developed. This methodology makes possible the identification of more than 20 species belonging to the families Carangidae, Mullidae, and Scombridae. The main novelty of this work lies in the longest number of different horse mackerel species included and in the applicability of the developed methods to all kinds of processed products that can be found by consumers in markets around the world, including those that have undergone intensive processes of transformation, as for instance canned foods. Finally, the methods were applied to 15 commercial samples, all of them canned products. Therefore, these methods are useful for checking the fulfillment of labeling regulations for horse mackerels and horse mackerel products, verifying the correct traceability in commercial trade, and fisheries control.     

Genetic identification of squids (families Ommastrephidae and Loliginidae) by PCR-RFLP and FINS methodologies

Cephalopods are a taxonomic group that contains a great number of families, genera and species, with many of them very important at the commercial level. The existence of very similar species in this class added up to the transformation process applied to them makes it difficult or even impossible for species identification based on morphological characterization. Moreover, the global commerce makes it possible that one determined species can be marketed in its antipodes. These questions suggest the necessity of molecular techniques to solve this situation. In the present work, a genetic method was developed on the basis of the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and forensically informative nucleotide sequencing (FINS) technique and makes possible the identification of more than 20 species belonging to the families Ommastrephidae and Loliginidae, as well as some octopus and sepia species. The PCR was employed to amplify 651 and 208 bp fragments of the mitochondrial cytochrome b gene. These molecular systems were applied to fresh, frozen, precooked, even canned cephalopods, allowing for the identification of the species included in these products. Therefore, these molecular tools could be applied in questions related to correct labeling, traceability, and importation controls of squids, sepias, and octopuses.     

Statistical validation of the identification of tuna species: bootstrap analysis of mitochondrial DNA sequences

Sequencing of the mitochondrial cytochrome b gene has been used to differentiate three tuna species: Thunnus albacares (yellowfin tuna), Thunnus obesus (bigeye tuna), and Katsuwonus pelamis (skipjack). A PCR amplified 528 bp fragment from 30 frozen samples and a 171 bp fragment from 26 canned samples of the three species were analyzed to determine the intraspecific variation and the positions with diagnostic value. Polymorphic sites between the species that did not present intraspecific variation were given a diagnostic value. The genetic distance between the sequences was calculated, and a phylogenetic tree was constructed, showing that the sequences belonging to the same species clustered together. The bootstrap test of confidence was used to determine the statistical validation of the species assignation, allowing for the first time a quantification of the certainty of the species assignation. The bootstrap values obtained from these results indicate that the sequencing of the cytochrome b fragments allows a correct species assignation with a probability > or =95%.     

Identification of Hake species (Merluccius Genus) using sequencing and PCR-RFLP analysis of mitochondrial DNA control region sequences.

The use of DNA-based methodologies in identification of hake species belonging to the Merluccius genus was shown to be successful. A short fragment of the left hypervariable domain of the mitochondrial control region was amplified, sequenced, and digested from 11 hake species. The hake-specific PCR product, due to its limited size, was obtained in a variety of tissue samples with different levels of DNA concentration and degradation, including sterilized food products. On the basis of this phylogenetically informative 156-bp sequence were selected four restriction enzymes (ApoI, DdeI, DraIII, and MboII) that allow the hake species discrimination. Species identification by phylogenetic analysis of sequences or by PCR-RFLP methodologies is useful in a variety of scenarios including authentication of thermally processed food, detection of food components, and species determination of individuals whose morphological characters are removed.     

Identification of anchovy (Engraulis encrasicholus L.) and gilt sardine (Sardinella aurita) by polymerase chain reaction, sequence of their mitochondrial cytochrome b gene, and restriction analysis of polymerase chain reaction products in semipreserves

A method of authenticating anchovy (Engraulis encrasicholus L.) and gilt sardine (Sardinella aurita) semipreserves (salt-cured and fillets in oil) has been developed by polymerase chain reaction (PCR) followed by sequence and restriction site analysis. The amplification of a fragment of the cytochrome b gene by universal primers produced a 376 base pairs (bp) fragment in all samples analyzed. Digestion of PCR products with XhoI, TaqI, AluI, and HinfI endonucleases yielded species-specific profiles distinguishing anchovy from gilt sardine. Therefore, the restriction length fragment polymorphism (RLFP) technique can be used to determine the species identity of anchovy and gilt sardine in semipreserves.     

Molecular phylogeny and species identification of sardines

The DNA sequence diversity of Sardina pilchardus (Walbaum, 1792) and some closely related species of Clupeomorpha was investigated using the mitochondrial DNA gene encoding cytochrome b. The nucleotide sequences of complete and partial mtDNA cytochrome b were determined in numerous specimens. Sequence divergence between species and genera was evenly distributed in the cytochrome b gene but rather high compared to reports for other fish species. Phylogenetic analyses on complete cytochrome b were used to study the relationships among the considered species. S. pilchardus was easily differentiated, showing a genetic distance of 0.25 with respect to Clupeidae species and 0.26 with respect to the other species. A species-specific short fragment (<150 bp) was isolated by polymerase chain reaction (PCR) using primers designed for Clupeomorpha. A rapid and reliable PCR method using restriction fragment length polymorphism (RFLP) with two restriction enzymes (MnlI/HinfI) was optimized for unambiguous differentiation of S. pilchardus from the other species tested (raw and canned products).     

Development of a DNA-based method aimed at identifying the fish species present in food products

Analysis of restriction fragment length polymorphism (RFLP) profiles of a 464 bp amplicon obtained from the mitochondrial cytochrome b gene was used to differentiate between several different fish species. The method was tested by a collaborative study in which 12 European laboratories participated to ascertain whether the method was reproducible. Each laboratory was required to identify 10 unknown samples by comparison with RFLP profiles from authentic species. From a total of 120 tests performed, unknown samples were correctly identified in 96% of cases. Further work attempting to use the method to analyze mixed and processed fish samples was also performed. In all cases the species contained within mixed samples were correctly identified, indicating the efficacy of the method for detecting fraudulent substitution of fish species in food products.     

PCR-RFLP analysis of mitochondrial DNA: a reliable method for species identification

A method for identification of game species has been developed on the basis of the amplification of a specific part of the mitochondrial genome (tRNA(Glu)/cytochrome b) using the polymerase chain reaction (PCR). To distinguish between several game species, the obtained 464-bp-long PCR products were cut with different restriction endonucleases (RE) resulting in species-specific restriction fragment length polymorphism (RFLP). Even closely related deer species could be distinguished by application of one or two RE. Natural polymorphisms of the target sequence within one species were examined for red deer (Cervus elaphus), and base pair substitutions were identified affecting the RFLP pattern.     

Application of denaturing high-performance liquid chromatography (DHPLC) for the identification of fish: a new way to determine the composition of processed food containing multiple species

The identification of fish species in transformed food products is difficult because the existing methods are not adapted to heat-processed products containing more than one species. Using a common to all vertebrates region of the cytochrome b gene, we have developed a denaturing high-performance liquid chromatography (DHPLC) fingerprinting method, which allowed us to identify most of the species in commercial crab sticks. Whole fish and fillets were used for the creation of a library of referent DHPLC profiles. Crab sticks generated complex DHPLC profiles in which the number of contained fish species can be estimated by the number of major fluorescence peaks. The identity of some of the species was predicted by comparison of the peaks with the referent profiles, and others were identified after collection of the peak fractions, reamplification, and sequencing. DHPLC appears to be a quick and efficient method to analyze the species composition of complex heat-processed fish products.     

Fish species identification in surimi-based products

Whole fish morphologically identified as belonging to Theragra chalcogramma, Merluccius merluccius, Merluccius hubbsi, and Merluccius capensis and 19 fish products commercialized as surimi with different commercial brands and labeled as T. chalcogramma were analyzed by direct sequence analysis of the cytochrome b gene. A phylogenetic analysis of surimi products was performed as well. Results demonstrated that mislabeling is a large-scale phenomenon, since 84.2% of surimi-based fish products sold as T. chalcogramma (16/19) were prepared with species different from the one declared. In fact, only three samples (samples 15-17) were found to belong to T. chalcogramma. In the remaining samples, Merluccidae (samples 4-14), Gadidae (samples 18 and 19), Sparidae (sample 1), and Pomacentridae (samples 2 and 3) families were detected. A phylogenetic tree was constructed, and the bootstrap value was calculated. According to this methodology, 11 samples were grouped in the same clade as Merluccius spp.     

Identification of gadoid species in fish meat by polymerase chain reaction (PCR) on genomic DNA

Identification of fish species is significant due to the increasing interest of consumers in the meat of sea fish. Methods focusing on fish species identification help to reveal fraudulent substitution among economically important gadoid species in commercial seafood products. The objective of this work was to develop a conventional PCR method for the differentiation of the following gadoid fish species in fish products: Alaska pollack ( Theragra chalcogramma), blue whiting ( Micromesistius poutassou), hake spp. ( Merluccius spp.), Atlantic cod ( Gadus morhua), saithe ( Pollachius virens), and whiting ( Merlangius merlangus). The species-specific primer pairs for gadoid species determination were based on the partial pantophysin I ( PanI) genomic sequence. Sequence identification was confirmed by cloning and sequencing of the PCR products obtained from the species considered. For the simultaneous detection of Alaska pollack, blue whiting, and hake spp., a quadruplex PCR system was constructed. Other gadoid species were detected in separate PCR reactions. After optimization of the reactions, the developed PCR systems were used for the analysis of codfish samples obtained from the Czech market and the customs' laboratories. This method represents an alternative approach in the use of genomic DNA for the identification of fish species. This method is rapid, simple, and reliable without the need for further confirmative methods. Furthermore, the identification of a mixture of more than one species is possible. The PCR system has been optimized for routine diagnostic purposes.     

Development of a method for the genetic identification of mussel species belonging to Mytilus, Perna, Aulacomya, and other genera

Legislation regarding the labeling of processed products is an important issue in the protection of consumer rights. This labeling is especially important in products that cannot be identified on the basis of their morphological characters, because these are removed from the animal in the transformation process. The goal of this study was the identification of mussel species using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) and Forensically Informative Nucleotide Sequencing (FINS) methodologies. The molecular marker selected was 18S rDNA (nuclear small-subunit rDNA gene), which allows identification at the genus level and at the species level in some cases. The genera included in this study were Mytilus, Perna, Aulacomya, Semimytilus, Brachidontes, Choromytilus, and Perumytilus. Different markers were used for genetic identification at the species level. To identify the species included in the genus Perna and Choromytilus, a fragment of ITS 1 (Internal Transcribed Spacer 1) was amplified by multiplex PCR and digested with restrictases. The species of Mytilus were identified by length polymorphism and RFLP of the polyphenolic adhesive protein gene. This methodology was validated with products manufactured in the authors' pilot plant and applied to commercial samples. Therefore, this sequential method can be completely or partially used to determine the mussel genus or species present in any food product.     

Use of restriction fragment length polymorphism to distinguish between salmon species

Identification of 10 salmon species using DNA-based methodology was investigated. Amplification of DNA was carried out using a primer set which amplified a region of the mitochondrial cytochrome b gene. Sequences of PCR-amplified DNA from the salmon species were used to select six restriction enzymes allowing species to be uniquely classified. RFLP patterns generated following analysis with each enzyme were resolved using polyacrylamide gel electrophoresis and visualized by silver staining. Results indicate that it is possible to differentiate between all 10 salmon species and that the technique could be easily adopted by the food industry for analysis of processed salmon products.     

Authentication of anglerfish species (Lophius spp) by means of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and forensically informative nucleotide sequencing (FINS) methodologies

Lophius represents the most important genus of the family Lophiidae from a commercial point of view. The main marketing formats of the species included in this genus are tails and cheeks, making impossible the species identification on the basis of their morphological characters. In the present study, two methods based on the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and phylogenetic analysis of DNA sequences [forensically informative nucleotide sequencing (FINS)] were developed to differentiate the seven species contained in the genus Lophius. In both cases, the molecular marker studied was the cytochrome oxidase subunit I gene (COI). The RFLP analysis of the PCR products digested with the endonuclease Mbo I generated species-specific restriction profiles, and the phylogenetic analysis showing a neighbor-joining tree with independent nodes was strongly supported for all of the studied species. These methods were applied to 40 commercial samples, allowing us to detect the samples incorrectly labeled. The fraudulent labeling ratio was higher in processed products (68.75%) than whole fish (31.25%). The species subjected to mislabeling were L. budegassa (68.75%), L. vomerinus (18.75%), and L. piscatorius (12.5%). Therefore, both methodologies can be independently used to authenticate the species belonging to the genus Lophius, being useful to check the fulfillment of labeling regulations of seafood products and to verify the correct traceability of commercial trade and the control of fisheries.