Analysis of raw meats and fats of pigs using polymerase chain reaction for Halal authentication

A method for species identification from pork and lard samples using polymerase chain reaction (PCR) analysis of a conserved region in the mitochondrial (mt) cytochrome b (cyt b) gene has been developed. Genomic DNA of pork and lard were extracted using Qiagen DNeasy^® Tissue Kits and subjected to PCR amplification targeting the mt cyt b gene. The genomic DNA from lard was found to be of good quality and produced clear PCR products on the amplification of the mt cyt b gene of approximately 360 base pairs. To distinguish between species, the amplified PCR products were cut with restriction enzyme BsaJI resulting in porcine-specific restriction fragment length polymorphisms (RFLP). The cyt b PCR-RFLP species identification assay yielded excellent results for identification of pig species. It is a potentially reliable technique for detection of pig meat and fat from other animals for Halal authentication.     

Development of a method for the identification of scombroid and common substitute species in seafood products by FINS

In the present work, a method for the authentication of scombroid products was developed, by means of FINS (Forensically Informative Nucleotide Sequencing) technique (Polymerase Chain Reaction (PCR) followed by phylogenetic analysis). The methodology developed allows the identification of most important scombroid species using the mitochondrial cytochrome b as molecular marker. Due to the different commercial value of the species belonging to this family, substitutions between species in seafood products can take place.     

Species authentication of octopus,cuttlefish, bobtail and bottle squids (families Octopodidae,Sepiidae and Sepiolidae) by FINS methodology in seafoods

In the present work a molecular a method for the authentication of cephalopods products was developed, which allows the genetic identification of about 30 species belonging to the families Octopodidae, Sepiidae and Sepiolidae. This molecular system is based on the phylogenetic analysis of DNA sequences. The molecular marker studied was the cytochrome b gene (cyt b), that was amplified by PCR and subsequently sequenced. The developed methodology was validated and further applied to 20 commercial samples, detecting 6 that were incorrectly labelled (30%). Therefore, this molecular tool could be applied in questions related to correct labelling, traceability, and import control of products containing the taxonomic groups studied.     

Detection of pig derivatives in food products for halal authentication by polymerase chain reaction –restriction fragment length polymorphism

A method for detection of the presence of pig derivatives in three types of food products—sausages and casings, bread and biscuits—using polymerase chain reaction–restriction fragment length polymorphism (PCR‐RFLP) analysis of a conserved region in the mitochondrial (mt) cytochrome b (cyt b) gene was developed. Genomic DNA of sausages and casings, bread and biscuits were extracted. The genomic DNA from the food products were found to be of good quality for the sausages and produced clear PCR products on the amplification of the mt cyt b gene of approximately 360 base pairs (bp). However, no genomic DNA was detected from the casing samples and poor quality of genomic DNA was extracted from bread and biscuits. No amplification of mt cyt b gene was produced from bread and biscuit samples. To differentiate between samples, the amplified PCR products were digested with restriction enzyme (RE) BsaJI, resulting in species‐specific RFLP. The cyt b PCR‐RFLP species identification assay gave excellent results for detection of pork adulteration in food products and is a potentially reliable technique to avoid species adulteration or fraudulent species substitution for halal authentication. Copyright © 2006 Society of Chemical Industry     

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.     

DNA‐Based Methods for the Identification of Commercial Fish and Seafood Species

ABSTRACT: The detection of species substitution has become an important topic within the food industry and there is a growing need for rapid, reliable, and reproducible tests to verify species in commercial fish and seafood products. Increases in international trade and global seafood consumption, along with fluctuations in the supply and demand of different fish and seafood species, have resulted in intentional product mislabeling. The effects of species substitution are far‐reaching and include economic fraud, health hazards, and illegal trade of protected species. To improve detection of commercial seafood fraud, a variety of DNA‐based techniques have been developed, including Multiplex PCR, FINS, PCR‐RFLP, PCR‐RAPD, PCR‐AFLP, and PCR‐SSCP, which are all based on polymorphisms in the genetic codes of different species. These techniques have been applied in the differentiation of many types of fish and seafood species, such as gadoids, salmonids, scombroids, and bivalves. Some emerging technologies in this field include the use of real‐time PCR, lab‐on‐a‐chip, and DNA microarray chips. In this review article, the major DNA‐based methods currently employed in the authentication of commercial fish and seafood species are discussed and future trends are highlighted. Examples of commercial applications and the use of online database resources are also considered.     

Meat species identification and Halal authentication analysis using mitochondrial DNA

A method utilizing PCR-restriction fragment length polymorphism (RFLP) in the mitochondrial genes was developed for beef (Bos taurus), pork (Sus scrofa), buffalo (Bubalus bubali), quail (Coturnix coturnix), chicken (Gallus gallus), goat (Capra hircus), rabbit (Oryctolagus cuniculus) species identification and Halal authentication. PCR products of 359-bp were successfully obtained from the cyt b gene of these six meats. AluI, BsaJI, RsaI, MseI, and BstUI enzymes were identified as potential restriction endonucleases to differentiate the meats. The genetic differences within the cyt b gene among the meat were successfully confirmed by PCR-RFLP. A reliable typing scheme of species which revealed the genetic differences among the species was developed.     

Detection of porcine DNA in gelatine and gelatine-containing processed food products-Halal/Kosher authentication

A commercially available real-time PCR, based on a multi-copy target cytochrome b (cyt b) using porcine specific primers, has been validated for the Halal/Kosher authentication of gelatine. Extraction and purification of DNA from gelatine were successfully achieved using the SureFood® PREP Animal system, and real-time PCR was carried out using SureFood® Animal ID Pork Sens kit. The minimum level of adulteration that could be detected was 1.0% w/w for marshmallows and gum drops. A small survey was undertaken of processed food products such as gum drops, marshmallows and Turkish delight, believed to contain gelatine. Of fourteen food products from Germany, two samples were found to contain porcine gelatine, whereas of twenty-nine samples from Turkey twenty-eight were negative. However, one product from Turkey contained porcine DNA and thus was not Halal, and neither was the use of porcine gelatine indicated on the product label.     

Authentication of raw and processed tuna from Indonesian markets using DNA barcoding,nuclear gene and character-based approach

Establishing seafood authentication methods is an important task for fisheries research laboratories and food control authorities. Nowadays, the extent of fish species substitution is suspected being greater than ever before in commercial markets. In order to provide reliable polymerase chain reaction (PCR)-based authentication systems for tunas, we collected and analyzed authentic tuna reference samples and tuna-food products from Indonesian markets. Our analytical methods mainly relied on identification using the mitochondrial cytochrome c oxidase subunit I (COI) gene, as a genetic marker for “DNA barcoding,” as well as the rhodopsin (RH1) gene as a nuclear marker. Additionally, we identified species-specific nucleotide diagnostic positions (characters) to complete the results obtained basic local alignment search (BLAST) and phylogenetic analysis. Authentication results of tuna-food products showed relatively successful amplification for the COI gene; RH1 acted as an alternative solution for some of the samples, which had failed to react in COI-PCR. Species of the genus Thunnus could not be unambiguously differentiated by BLAST and phylogenetic analysis (neighbor-joining tree) in all cases due to the high similarity of the COI sequences. However, the character-based identification method was found to be helpful for species assignment in case of tuna-food products. Therefore, our findings demonstrated that the COI gene could be more reliable used as a tool for Indonesian commercial tuna products authentication, if the sequencing results were combined with the character-based identification using differences at certain nucleotide positions.     

A multiplex-PCR assay for the authentication of mackerels of the genus Scomber in processed fish products

In this study, we have developed a novel multiplex-PCR assay for the authentication of mackerels of the genus Scomber in processed food. The method consists of two novel Scomber japonicus- (104 bp) and Scomber australasicus-specific (143 bp) amplicons, respectively, corresponding to the mitochondrial control region. It also includes the previously described Scomber colias-specific product (159 bp) corresponding to the 5S ribosomal DNA, the Scomber scombrus-specific fragment (123 bp) from the mitochondrial NADH dehydrogenase subunit 5, and finally a positive amplification control corresponding to the small 12S rRNA subunit (188 bp). The system was assayed in fresh samples as well as in a total of 40 commercial samples including 28 different canned products and 12 unprocessed fresh fillets. A positive identification was observed in all cases according to their commercial labelling. Overall, this methodology reveals as a potential molecular tool for direct application in the authentication of Scomber mackerels in the seafood industry.     

Molecular identification of Korean ginseng by amplification refractory mutation system-PCR

Expensive herbs like ginseng are always a possible target for fraudulent labeling. Korean ginseng (Panax ginseng) is among the most popular and expensive plant species in many countries, including China, Japan, Korea, Russia, Canada, and the USA. Polymerase chain reaction (PCR) analysis of the internal transcribed spacer (ITS) has proven an appropriate method for the identification of the most popular species (P. ginseng) in the Panax ginseng genus (Panax). A single nucleotide polymorphism (SNP) has been identified between P. ginseng and other Panax species. Specific PCR primers were designed from this SNP site within the sequence data, and were used to detect Korean ginseng (P. ginseng) via multiplex PCR. The established multiplex-PCR method for the simultaneous detection of Korean ginseng and foreign ginseng in a single reaction was determined to be effective. The described method has important implications in both the production and sale of commercial ginseng products, allowing for the prevention of fraud, and also revealing the possible presence of other, cheaper ginseng species.     

FINS methodology to identification of sardines and related species in canned products and detection of mixture by means of SNP analysis systems

The sardines are a resource of great importance in the artisanal and industrial fisheries worldwide. Such sardines and sardine-type products are included many species of small pelagic species, all belonging to the Clupeidae family. Within this family, highlights the European sardine (Sardina pilchardus), which by its organoleptic characteristics this species has gained an extraordinary commercial significance. In this work, an amplification of a fragment of mitochondrial cytochrome b marker and subsequent phylogenetic analysis (FINS: Forensically Informative Nucleotide Sequencing) were carried out to assure the correct labeling of sardine and sardine-type products. On the other hand, a single nucleotide polymorphism (SNP) analysis that allows detection of mixture of S. pilchardus and S. aurita in canned products was developed. After the application of this methodology to more than 80 available commercial samples can conclude that in more of 15% of the products analyzed, the name of the species displayed in the label was not in agreement with the identified species, also a one of these included in mixture of species. The main novelty of this work lies in the fact that have been included a long number of different species of sardines, many of which were not included in similar works up to date. Furthermore, this methodology allows identifying over 20 species of sardines and can be applied to all kinds of processed products, including those who have been subjected to intensive processing treatments, such as canned foods. Therefore, this molecular tool can be applied in questions related to correct labeling, traceability, fishery regulations, and commercial trade control of sardines and sardine-type products.     

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 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.     

Consumer willingness to pay for quality attributes of fresh seafood: A labeled latent class model

We applied a labeled choice experiment (LCE) to investigate consumer demand and choice behavior for fresh seafood in a retail market. The LCE was conducted for a large number of seafood alternatives (i.e., seafood species) labeled by the respective seafood name (e.g., cod, salmon, mussels). Consumer heterogeneity in preference was expressed by estimating a labeled latent class model with alternative-specific effects, which varies choice probability and model parameters over seafood alternatives and across classes. The willingness to pay (WTP) for extrinsic attributes (e.g., product form, production method, and country of origin), and the rank ordered-intrinsic value were estimated for each seafood alternative within classes and the entire market. The WTP estimate in our study is expected to be more accurate than those derived from studies based on single product alternatives because the LCE allows respondents to evaluate choice alternatives through both attribute judgment and alternative comparison. Exploring a variety of product alternatives is also meaningful to firms with multiple products (e.g., fresh seafood retailers) or firms with many direct competitors.     

Detection of roe deer,red deer,and hare meat in raw materials and processed products available in Poland

To allow detection of meat from the most popular game species in Poland, we developed a PCR-based method for identification of roe deer (Capreolus capreolus), red deer (Cervus elaphus), and hare (Lepus europaeus). The designed primers were based on the noncoding, mitochondrial D-loop region. Amplicon sizes ranged from 116 to 255 bp. The primers exhibited no cross-reactivity with the DNA from common slaughter and other game species. The detection limit of the assay was established to be below 0.001 % in raw red deer (C. elaphus) and hare (L. europaeus) meat, and below 0.01 % in raw roe deer (C. capreolus) meat, whereas <0.5 % of hare and red deer meat in processed samples could be detected. The PCR-based assay was used for authentication of 17 samples of raw game meat and 32 samples of game meat-containing products available in Polish markets. Analysis of all tested raw meat and processed products revealed the presence of DNA of investigated species in concordance with producers’ declarations.     

Vibrio species involved in seafood-borne outbreaks (Vibrio cholerae,V. parahaemolyticus and V. vulnificus): Review of microbiological versus recent molecular detection methods in seafood products

Seafood products are widely consumed all around the world and play a significant role on the economic market. Bacteria of the Vibrio genus can contaminate seafood and thus pose a risk to human health. Three main Vibrio species, V. cholerae, V. parahaemolyticus and V. vulnificus, are potentially pathogenic to humans. These species are responsible for a dramatic increase of seafood-borne infections worldwide. Hence, early detection of total and pathogenic Vibrio is needed and should rely on quick and effective methods. This review aims to present the standard methods FDA-BAM, ISO/TS 21872–1:2007 and TS 21872–2:2007 and compare them to recent molecular biology methods including endpoint PCR, quantitative real-time PCR (qPCR) and PCR-derived methods with a focus on LAMP (loop-mediated isothermal amplification). The available methods presented here are dedicated to the detection and identification of the Vibrio species of interest in seafood.     

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.     

Detection and differentiation of Vibrio spp. in seafood and fish samples with cultural and molecular methods

Vibrio spp. as natural inhabitants of sea- and brackwater of both tropical and temperate regions of the world are commonly found in different kinds of seafood. Even among the three main human pathogenic species Vibrio parahaemolyticus, Vibrio cholerae and Vibrio vulnificus most of the isolates from seafood do not carry the different virulence factors responsible for foodborne infections. Therefore, the risk assessment of Vibrio spp. in seafood is currently based mainly on the knowledge of the genetic setting of foodborne strains. For the detection and differentiation of Vibrio spp. (V. parahaemolyticus, V. cholerae and V. vulnificus) three probe-based multiplex real-time PCR systems were developed and validated. One real-time PCR system simultaneously detects V. parahaemolyticus, V. cholerae and V. vulnificus on genus level combined with an Internal Amplification Control. The detection limit for the system was between 1 cfu/mL and 10 cfu/mL in pure culture and in different artificially contaminated sample material, e. g. prawns or Alaska Pollock. The other two PCR systems were implemented for the detection of different virulence genes of V. parahaemolyticus and V. cholerae isolates. The molecular detection systems were applied for the investigation of 338 raw and cooked seafood and fish samples for the presence of the different Vibrio spp. The collected data indicate that the PCR systems can be useful for rapid detection and differentiation of Vibrio spp. in different food matrices as basis for a preventive consumer protection policy.     

Genetic authentication by RFLP versus ARMS? The case of Moldavian dragonhead (Dracocephalum moldavica L.)

Moldavian dragonhead (Dracocephalum moldavica L.), due to its pleasant lemon scent and medical effects, has acquired increasing impact as functional food. The high diversity within the genus, limited supply not keeping pace with the growing demand, the morphological similarity with other Labiatae, and trading under the common name Turkish Melissa invite adulteration by surrogate species. We have developed several verified reference accessions of D. moldavica L. along with potential surrogate species to compare different approaches of authentication, also in commercial samples. We report on three strategies of authentication—a microscopic method, based on the relative size of epidermal pavement cells versus palisade cells, and two strategies of genetic authentication based on the barcoding marker large subunit of ribulose-1,5-bisphosphate carboxylase oxygenase (rbcL). We can detect single-nucleotide exchanges between D. moldavica L. and the potential surrogate species Melissa officinalis L. and Nepeta cataria L. by restriction fragment length polymorphism (RFLP), and we show that we can use this to verify the presence of D. moldavica even in dried and highly fragmented mixtures from commercial samples. We further develop a third strategy derived from the so-called amplification refractory mutation system (ARMS), based on multiplex PCR of the rbcL marker upon addition of specifically designed intermediate primers that will generate a diagnostic second band in case of D. moldavica L., but not for the surrogate species. We demonstrate that this ARMS approach is superior to the RFLP strategy, because it safeguards against experimental fluctuations, can unequivocally verify the presence of D. moldavica in commercial samples declaring its presence, and yields a clear outcome in a one-step protocol.