Application of DNA‐Based Methods to Identify Fish and Seafood Substitution on the Commercial Market

ABSTRACT: Fish and seafood substitution has become an important concern in domestic and international marketplaces, in part due to increased international trade, per capita seafood consumption, and production of processed foods. In many cases, seafood substitution is a form of economic deception, where highly prized species are substituted with those of lesser value. To prevent illegal species substitution, a number of DNA‐based methods have been developed to detect fish and seafood species in commercial products. These methods, along with common gene targets, have been reviewed previously in this journal. The current article is meant to build upon earlier discussions by providing a comprehensive review of the application of these DNA‐based methods to the discovery of fish and seafood substitution on the commercial market. Popular food uses, potential substitution cases, and peer‐reviewed research articles published to date are discussed for all major species groups of concern, including flatfish, gadoids, scombroids, salmonids, percoids, sturgeons, sharks, eels, and bivalves. The use of DNA‐based methods to monitor commercial whale meat products is also reviewed.     

Development of a genetic method for the identification of salmon,trout, and bream in seafood products by means of PCR–RFLP and FINS methodologies

In the present study, two alternative methods for identifying 13 salmon, trout and bream species were developed. Both of them are based on polymerase chain reaction (PCR) amplification of a cytochrome b gene fragment. Subsequently, different techniques were assayed to assign the PCR amplicons previously obtained to particular species. The first one is based on the restriction fragment length polymorphism (RFLP) and includes three endonucleases for generating species-specific restriction profiles, while the second one is based on the phylogenetic analysis of DNA sequences. The main novelty of this work lies in the applicability of the developed methods to all kinds of processed products, including those undergoing intensive processes of transformation, as for instance canned foods. Finally, the methods were applied to 25 commercial samples including some that had been subjected to intensive thermal treatment, allowing the detection of those incorrectly labeled (16%). Therefore, these methods are useful to check the fulfillment of labeling regulations for seafood products, verify the correct traceability in commercial trade, and for fisheries control.     

Implementation of quality control methods (physico-chemical, microbiological and sensory) in conjunction with multivariate analysis towards fish authenticity

Nowadays authenticity of foods and fish in particular has become of crucial importance because of high number of adulteration cases. Authenticity control has gained ground thanks to the development of several rapid physico‐chemical and microbiological methods aiming at distinguishing one species from another based on solid scientific evidence. It has been proven that despite the precision and accuracy of robust analytical and protein and DNA‐based techniques, detection of authenticity could not be claimed without resorting to multivariate analysis. This review summarizes both the most advanced and state of the art used techniques for detecting fish and seafood authenticity (both in terms of species and geographical origin). Another issue reported in this review is the preservation of fish and seafood through the implementation of old and novel techniques (ice, modified atmosphere packaging). Several informative tables were included in this paper referring to the employed quality control and sensory analysis methods and multivariate analysis for fish and seafood.     

The use of molecular markers in the verification of fish and seafood authenticity and the detection of adulteration

The verification of authenticity and detection of food mislabeling are elements that have been of high importance for centuries. During the last few decades there has been an increasing consumer demand for the verification of food identity and the implementation of stricter controls around these matters. Fish and seafood are among the most easily adulterated foodstuffs mainly due to the significant alterations of the species' morphological characteristics that occur during the different types of processing, which render the visual identification of the animals impossible. Even simple processes, such as filleting remove very important morphological elements and suffice to prevent the visual identification of species in marketed products. Novel techniques have therefore been developed that allow identification of species, the differentiation between species and also the differentiation of individuals that belong to the same species but grow in different populations and regions. Molecular markers have been used during the last few decades to fulfill this purpose and several improvements have been implemented rendering their use applicable to a commercial scale. The reliability, accuracy, reproducibility, and time-and cost-effectiveness of these techniques allowed them to be established as routine methods in the industry and research institutes. This review article aims at presenting the most important molecular markers used for the authentication of fish and seafood. The most important techniques are described, and the results of numerous studies are outlined and discussed, allowing interested parties to easily access and compare information about several techniques and fish/seafood species.     

Rapid quantitative detection of, Listeria monocytogenes in salmon products: evaluation of pre-real-time PCR strategies

The spread and persistence of Listeria monocytogenes in smoked fish products and seafood processing factories are big concerns. Thus, the corresponding quality assurance programs must include adequate microbiological control measures. We evaluated eight different pre-PCR sample processing strategies to be coupled with a previously developed real-time PCR assay for the quantitative detection of L. monocytogenes in salmon products. The optimal pre-PCR procedure involved filtration and DNA purification with the use of a commercial kit. This strategy could detect 10 CFU of L. monocytogenes per g of smoked salmon and could quantify 1,000 CFU/g with excellent accuracy compared with the standard plate count method. Thus, this method could be a promising alternative for the quantitative detection of L. monocytogenes in smoked fish products and processing factories. This method could also detect the bacterium in raw salmon.     

主要进出口经济鱼类及其制品分子鉴定技术的研究进展

随着我国进出口贸易的不断发展,进出口鱼类品种繁多,数量巨大。但部分国内外企业受高额利润驱使,以假充真、以劣充好事件时有发生,鱼类制品更是真伪难辨,掺假状况极其普遍。通过形态学和生物学方法已经不能满足对鱼类种属鉴定。近年来,随着分子生物技术的迅猛发展,分子检测技术在鱼类品种鉴定领域得到广泛应用。本文针对不同分子鉴定技术进行阐述,包括普通聚合酶链反应(polymerase chain reaction,PCR)技术、多重PCR技术、实时定量PCR技术、DNA条形码技术、环介导等温扩增检测技术(loop-mediated isothermal amplification,LAMP)、聚合酶链反应-限制性酶切多态性技术(polymerase chain reaction-restriction fragment length polymorphism,PCR-RFLP)以及随机扩增多态性DNA检测技术(random amplified polymorphic DNA),并根据不同检测技术的特点进行分析,为进出口鱼类鉴定提供技术支撑。     

A method to detect the parasitic nematodes from the family Anisakidae, in Sardina pilchardus, using specific primers of 18 S DNA gene

Anisakiasis is the infestation of man by the third larval stage of the Anisakidae family parasites, caused by the consumption of raw or undercooked seafood. The hosts of the third larvae parasite are marine fishes, among which Sardina pilchardus is of high commercial interest in many countries. So, the detection of anisakid larvae in fisheries products (fresh and processed) is of great interest to a rapid and feasible diagnosis of parasitism. Currently, there are different types of parasite detection methods but they are all difficult and tedious. The aim of this work was to detect, using DNA-based techniques, the presence of anisakid larvae in the entire fish host. Three pairs of primers were designed in the 18 S DNA gene taking into account the maximum matching between the anisakid species sequences and the minimum matching concerning for both the fish species and some other groups of organisms that parasitize fish (non-anisakid nematodes, trematodes, and cestodes). Experimental mixtures of parasite and fish host were made and the sensitivity of anisakid DNA detection tested by PCR. All the above primers and two others combinations of them were specific for the nematode group within the studied species, and the sensitivity of detection was on the order of 1 part of nematode to 100 000 parts of fish (anisakid weight:fish sample fresh weight = 1:≈100 000).     

Universal mitochondrial 16s rRNA biomarker for mini-barcode to identify fish species in Malaysian fish products

Mislabelling in fish products is a highly significant emerging issue in world fish trade in terms of health and economic concerns. DNA barcoding is an efficient sequencing-based tool for detecting fish species substitution but due to DNA degradation, it is in many cases difficult to amplify PCR products of the full-length barcode marker (~650 bp), especially in severely processed products. In the present study, a pair of universal primers targeting a 198 bp sequence of the mitochondrial 16s rRNA gene was designed for identification of fish species in the processed fish products commonly consumed in Malaysia. The specificity of the universal primers was tested by both in-silico studies using bioinformatics software and through cross-reaction assessment by practical PCR experiments against the DNA from 38 fish species and 22 other non-target species (animals and plants) and found to be specific for all the tested fish species. To eliminate the possibility of any false-negative detection, eukaryotic endogenous control was used during specificity evaluation. The developed primer set was validated with various heat-treated (boiled, autoclaved and microwaved) fish samples and was found to show high stability under all processing conditions. The newly developed marker successfully identified 92% of the tested commercial fish products with 96–100% sequence similarities. This study reveals a considerable degree of species mislabelling (20.8%); 5 out of 24 fish products were found to be mislabelled. The new marker developed in this work is a reliable tool to identify fish species even in highly processed products and might be useful in detecting fish species substitution thus protecting consumers’ health and economic interests.     

Validation of a method for the detection of five species,serogroups, biotypes and virulence factors of Vibrio by multiplex PCR in fish and seafood

In this work a sequential multiplex PCR system was designed and validated for the detection of most frequent foodborne pathogen Vibrio species in fish and seafood (Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio alginoliticus and Vibrio mimicus). The method proposed functions in a hierarchical way, being composed of an end-point multiplex PCR to detect the presence of DNA belonging to the studied species, followed by multiplex PCR and fragment analysis allowing the viability assessment of the detected strains. The final multiplex PCR step of the method may be applied if identification of the serogroup, biotype and/or virulence factor level is necessary. Forty samples of commercial fish and seafood products were used at the method validation stage. Sixty three marine organism samples obtained from various estuarine areas of Spain including shrimps, crabs, bivalve mollusks and fishes were screened for presence of Vibrio species and 2 mussel samples were found positive for V. parahaemolyticus. On the whole, the proposed method is robust and readily adaptable in routine molecular diagnostic laboratories, allowing monitoring and simultaneous detection of all these bacterial pathogens in seafood samples, reducing the expenses and time consumed by other analytical methods.     

Authentication of the most important species of rockfish by means of fins

In the present work, a method for the authentication of more of 100 scorpaenids species has been developed by means of forensically informative nucleotide sequencing technique, which is based on a PCR followed by a phylogenetic analysis. Due to the different commercial value of the species belonging to this taxonomic group, substitutions between species in seafood products can take place. Two different methodological strategies are proposed, allowing the analysis of fresh, frozen, or highly processed products with total reliability. These analytical systems have been validated and subsequently applied to 25 commercial samples. Therefore, this technique can be used as a routine method to avoid the mislabeling in the marketing of scorpaenid species and it is also suitable to assess the correct seafood traceability of these products.     

Detection of Different DNA Animal Species in Commercial Candy Products

Candy products are consumed all across the world, but there is not much information about their composition. In this study we have used a DNA‐based approach for determining the animal species occurring in 40 commercial candies of different types. We extracted DNA and performed PCR amplification, cloning and sequencing for obtaining species‐informative DNA sequences. Eight species were identified including fish (hake and anchovy) in 22% of the products analyzed. Bovine and porcine were the most abundant appearing in 27 samples each one. Most products contained a mixture of species. Marshmallows (7), jelly‐types, and gummies (20) contained a significantly higher number of species than hard candies (9). We demonstrated the presence of DNA animal species in candy product which allow consumers to make choices and prevent allergic reaction.     

POTENTIAL FUTURE CHALLENGES TO THE APPLICATION OF GAMMA IRRADIATION TO SEAFOOD IN TUNISIA

There is an increasing consumer demand for high quality, minimally processed, additive-free and microbiologically safe foods. The future application of irradiation to the seafood industry is developing. In order to investigate the feasibility of using gamma irradiation in the fish and seafood industry, questionnaires were distributed to the managers of 25 fish exporting companies. The cost of irradiation and the additional transport services were discussed and mostly accepted. This survey showed that essential conditions are to be found in Tunisia to introduce irradiation technology into the exporting seafood industry. However, the legal status of seafood irradiation varies in some importing countries (mainly European Union [EU]). Regulators within the EU have taken different approaches to allowing processors to use this technology. Such inconsistency is related to the objection of misinformed consumers. Consequently, commercial trade of irradiated seafood could be impeded.

PRACTICAL APPLICATIONS

This article provides evidence of the importance of industry managers' role in the food chain when testing the feasibility of a new process. Fish shelf-life extension using gamma irradiation is an emerging technology that presents many advantages to all food chain components and allows international trade requirements to be met. The seafood export industry in Tunisia shows the technological need for such a process. Furthermore, this study demonstrated that the economic cost of irradiation in Tunisia is acceptable and that it could enhance fish export capacity. However, the legal status of fish irradiation in the import countries, mainly the European Union, should be modernized in order to assure the feasibility of irradiated fish export from Tunisia.     

Mitochondrial cytochrome b DNA sequence variations: an approach to fish species identification in processed fish products

The identification of fish species in food products is problematic because morphological features of the fish are partially or completely lost during processing. It is important to determine fish origin because of the increasing international seafood trade and because European Community Regulation 104/2000 requires that the products be labeled correctly. Sequence analysis of PCR products from a conserved region of the cytochrome b gene was used to identity fish species belonging to the families Gadidae and Merluccidae in 18 different processed fish products. This method allowed the identification of fish species in all samples. Fish in all of the examined products belonged to these two families, with the exception of one sample of smoked baccalà (salt cod), which was not included in the Gadidae cluster.     

The use of stable isotopes for authentication of gadoid fish species

The rise of processed seafood in international trade has increased the feasibility of fish species substitution. Gadidae fish species are sold commercially as salted fish, and differences in price between fish of different species may lead to falsification. The present study addresses this falsification issue by attempting to discriminate among salted Atlantic cod and salted saithe using isotope ratio mass spectrometry (IRMS) as well as the stable isotope ratios of carbon (δ¹³C) and nitrogen (δ¹⁵N). δ¹⁵N in tissues with lower turnover rates (bone and skin) and in tissues with greater turnover rates (muscle) can be used to authenticate the species of salted fish samples when distinguishing between Atlantic cod and saithe.     

Detection of Salmonella spp. in tropical seafood by polymerase chain reaction

The incidence of Salmonella spp. in tropical seafood was studied using standard microbiological techniques and polymerase chain reaction (PCR). Six of 20 finfish (30%), 4 of 20 clams (20%) and 1 of 20 shrimp (5%) were positive by culture techniques and by PCR. In a comparative study of different selective enrichment broths and selective plating media, more than one enrichment broth and selective agar were found to be necessary for efficient detection of Salmonella from seafood. Selenite cystine broth (SCB) was found to be more efficient compared to tetrathionate broth (TTB) while both bismuth sulfite agar (BSA) and hektoen enteric agar (HEA) were equally effective as selective plating media for fish. In the case of clams, HEA was found to be more effective. The presence of Salmonella spp. could be detected by PCR amplification of DNA extracted directly from the enrichment broths. In two cases, enrichment broths that were positive by PCR did not yield Salmonella by conventional methods.     

Methicillin‐Resistant Staphylococcus aureus in Seafood: Prevalence,Laboratory Detection,Clonal Nature,and Control in Seafood Chain

Methicillin‐resistant Staphylococcus aureus (MRSA), a versatile pathogen bearing multiple virulence determinants, is increasingly being detected in various food‐producing animals, including fish. In addition, it is a potential food poisoning agent. MRSA is not an inherent microbiota of fish; its presence is attributed to pre‐ or postharvest contamination through fish handlers, water, ice, and processing equipment. Several reviews have been written on MRSA in clinical as well as the food animal‐producing sector, but information specific to MRSA in seafood is scant. This review puts forth insights on MRSA detection in seafood, antibiotic resistance, diversity of clones in seafood, and possible control measures in seafood production chain. Emphasis has been given on assessing the variations in the protocols employed for isolation and identification in different food matrices and lay the foundation for researchers to develop optimized procedure.     

Phenolic Compounds and Plant Phenolic Extracts as Natural Antioxidants in Prevention of Lipid Oxidation in Seafood: A Detailed Review

Lipid oxidation is the principal cause of quality loss in seafood, which is known to contain high amounts of polyunsaturated fatty acids. Such quality deterioration, associated with the development of off‐flavor as well as lowering of nutritive value, can be retarded by incorporation of additives having antioxidative properties. The use of synthetic antioxidants has long been practiced in retarding lipid oxidation. However, due to the potential health concerns of synthetic antioxidants, polyphenolic compounds, which are found in different plants and their manufactured by‐products, have been used as an alternative natural antioxidants to retard lipid oxidation in different seafood systems. Both pure phenolic compounds and crude plant phenolic extracts have been successfully used in delaying oxidation in fish muscle, fish oil, and fish oil‐in‐water emulsions. This article reviews in detail the phenolic antioxidants and their natural sources as well as focuses on the role of pure phenolic compounds and crude plant phenolic extracts on the prevention of lipid oxidation in different seafood systems.     

Authentication of swordfish (<Emphasis Type="Italic">Xiphias gladius</Emphasis>) by RT–PCR and FINS methodologies

In the present study, two methods for the authentication of swordfish (Xiphias gladius) were developed. The first one is based on a TaqMan probe real-time PCR technology using the cytochrome oxidase subunit I (COI), while the second one is based on the phylogenetic analysis of DNA sequences (forensically informative nucleotide sequencing, FINS) using the cytochrome b (cyt b) gene fragment. Both techniques can be applied depending on the laboratory equipment and allow the detection of fraudulent or unintentional mislabelling of this species. The developed methodologies were validated and subsequently were applied to 30 commercial samples labelled as swordfish or X. gladius in order to determinate whether the species used for their manufacturing corresponded to this species. These tools are useful to clarify questions related to the correct labelling of commercial products and to verify the correct traceability in commercial trade and for fisheries control.     

Authentication of the most important species of freshwater eels by means of FINS

Eels are a taxonomic group with great commercial importance due to their huge steaks. They have very high demand, especially the young ones. There are high morphological similarity and different market values between different species. For these reasons arises the need to develop techniques that allow identifying as many species as possible. In this study, a DNA method based on DNA phylogenetic analysis of sequences (forensically informative nucleotide sequencing) has been developed. This method has been used to authenticate 12 eel species, including the most important to commercial level (Anguilla anguilla, A. rostrata, A. japonica, A. australis), by means of the amplification of a 239-base pair (bp) fragment of the mitochondrial Cytochrome b (cyt b) gene. This method is useful to clarify questions related to the correct labeling of commercial products and to verify the traceability in commercial trade and for fisheries control.     

Identification of fish species by 5S rRNA gene amplification

The 5S ribosomal RNA is a very suitable target for easy, rapid and inexpensive fish species identification due to its structure, consisting of a conserved region followed by a species-specific noncoding region called ‘nontranscribed spacer’. We have exploited this species-specificity in length and sequence to discriminate among fish species which can be subjected to substitution in the fish markets. After sequencing and alignment of the corresponding portions of the 5S rRNAs of different fish species, we have designed the primer pairs necessary for PCR amplification on the DNA traits which most diverged and a primer pair on conserved regions. Our results have shown the feasibility, simplicity and reliability of the proposed approach for the detection of mislabelling or fraudulent substitution of fish species.