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From: Food Quality & Safety magazine, June/July 2010

Fish and Chips

Lab-on-a-chip technology is one tool for fighting seafood substitution

by Paul Zavitsanos

An increasing number of recalls and cases involving adulteration of products such as infant formula, peanuts, and salami have eroded consumer confidence and put product fraud in the media spotlight. According to a study by the Grocery Manufacturers Association (GMA) and the GMA Science and Education Foundation, food product fraud may cost the food industry $10 to $15 billion per year. The melamine contamination of milk products, which cost the industry $10 billion and affected almost 300,000 consumers around the world, illustrated the global market consequences of product fraud.

Product fraud can be classified as either counterfeiting or economically motivated adulteration (EMA). While counterfeiting is the unauthorized representation of a registered trademark with the intent to deceive the purchaser, EMA is defined as the intentional fraudulent modification of a finished product for economic gain. Such adulteration manifests itself in many ways, including substitution of a lesser value ingredient.

Perhaps the most pervasive and widely publicized example of EMA by substitution is the replacement of high-value species of fish with those of low value (see “Fishy Business” on p. 12 of this issue). Increased international trade, rising worldwide fish and seafood consumption, and varying levels of supply and demand of certain species drive this practice.1 The Internet is rife with news reports of restaurants and fish markets serving fish that turn out to be a different species than the one advertised.

Red snapper is one type of fish that is often replaced with a lesser-value species. One investigation found that the red snapper sushi served at 14 different restaurants was actually a different species at all 14. Another examination found that of eight red snapper sushi samples tested, six were tilapia, a less expensive fish, and one was white bass. Only one was red snapper. Testing of red snapper at four restaurants and 10 grocery stores found that half of the restaurant samples and six of the grocery store samples were mislabeled.

A study by Therion International, a company that provides testing to determine the genetic identity of animals, determined that 19 of 39 restaurant samples were a less expensive species than red snapper, including tilapia and even horse mackerel. In fact, a survey by the National Marine Fisheries Service National Seafood Inspection Laboratory (NSIL) found that 80% of red snapper was mislabeled. The problem is not limited to red snapper, and it is not new; an NSIL study conducted from 1988 through 1997 found that 34% of tested seafood products were mislabeled (http://fshn.ifas.ufl.edu/seafood/sst/22ndAnn/file08.pdf).

The fish substitution problem is a serious one. In addition to the financial damage and loss of confidence it wreaks on the commercial market, it also puts consumers at risk of purchasing potentially harmful products and reduces the effectiveness of marine conservation and management programs that help protect ocean habitats and endangered species. Any fish species that appears to be readily available in the marketplace can create the public impression that there is a plentiful supply of that fish in the sea, but this perception may belie the true state of the fisheries’ stock.

In fact, a survey by the National Marine Fisheries Service National Seafood Inspection Laboratory (NSIL) found that 80% of red snapper was mislabeled. The problem is not limited to red snapper, and it is not new; an NSIL study conducted from 1988 through 1997 found that 34% of tested seafood products were mislabeled.

Industry Fights Fraud

The seafood industry plays a role in detecting and preventing seafood fraud because maintaining consumer confidence in seafood products is essential. Economic fraud in the industry, of which EMA is just one piece, erodes consumer trust in the product category, creates unfair economic advantages for companies willing to perform illegal acts, and promotes success through rule-breaking. Seafood industry associations represent various aspects of the industry throughout the supply chain, from harvesters to retailers, and include associations such as the National Restaurant Association and the National Fisheries Institute (NFI).

These groups work to protect the brand names and quality of their respective products or businesses; monitor issues and legislation that may impact the industry, including fraud and other illegal activities; and work with government agencies and other organizations to promote the health and viability of their industry. The NFI also created the Better Seafood Board to help its members combat such seafood fraud problems as transshipping to avoid antidumping duties, short-weighting, and species substitution. As a condition of membership, all members of NFI have pledged to abide by fair and lawful business practices.

Regulatory organizations like the Directorate-General for Maritime Affairs and Fisheries of the European Commission (EC) have established labeling laws for fish and aquaculture products requiring traceability information such as species identification, origin of fish, and production method. The EC adopted a regulation to prevent, deter, and eliminate illegal, unreported, and unregulated fishing, which went into effect on January 1, 2010. This regulation will have a significant impact on countries that trade in fisheries products with, but are not members of, the European Union (EU), because all fish materials imported into the EU after that date must be accompanied by catch certificates.

Seafood substitution is prohibited in the United States according to the Federal Food Drug and Cosmetic Act Section 403(b): Misbranded Food. Federal, state, and local agencies play a role in detecting and preventing seafood fraud throughout the supply chain. In general, federal agencies inspect seafood processors, distributors, importers, and imported seafood products. States also inspect seafood processors, either through contracts with the United States Department of Agriculture or under their own authority, and states and local governments inspect and regulate retail establishments such as restaurants and supermarkets.

Lab-on-a-chip technology is one of the newer tools for detecting mislabeled seafood products.
Lab-on-a-chip technology is one of the newer tools for detecting mislabeled seafood products.

Tools for Detection

Determination of seafood species has traditionally been based on species-specific electrophoretic, chromatographic, or immunological characteristics of proteins.1 Analyses have been done using isoelectric focusing, sodium dodecyl sulfate–polyacrylamide gel electrophoresis, capillary electrophoresis (CE), high-performance liquid chromatography, and immunoassay systems.

These methods are generally reliable for use with fresh or frozen tissue, but intense heat processing or drying can destroy the biochemical properties and structural integrity of proteins, often making protein analysis impractical. Enzyme-linked immunosorbent assay, which may be useful even in heat-sterilized products, has been used for the identification of several fish species. Immunoassays can be ineffective at differentiating closely related species, however, and they require the development of an antibody against the specific protein of interest.

The use of DNA-based methods for species detection offers a number of advantages over protein-based methods, including increased ease of use, specificity, sensitivity, and reliable performance with highly processed samples. DNA molecules are more thermostable than proteins during processing, and DNA fragments suitable for analysis can still be recovered following sterilization. As a result, numerous genetic methods are currently being used to identify certain fish and seafood species.

Some DNA-based methods employ polymerase chain reaction in combination with restriction fragment length polymorphism (PCR-RFLP), while others use sequencing through DNA barcoding or forensically informative nucleotide sequencing (FINS), amplified fragment length polymorphism, or single-strand conformation polymorphism. These techniques have been applied to the identification of numerous species of seafood, including gadoids, flatfish, salmonids, scombroids, sardines and anchovies, eels, mollusks, and many more.

Schematic diagram of the Agilent Technologies lab-on-a-chip technology used to perform fish species identification utilizing polymerase chain reaction-restriction fragment length polymorphism.
click for large version
Schematic diagram of the Agilent Technologies lab-on-a-chip technology used to perform fish species identification utilizing polymerase chain reaction-restriction fragment length polymorphism.

Detecting Species-Specific Variations

A popular method for DNA-based seafood identification is PCR-RFLP, which is based on variations in the lengths of particular restriction fragments generated from specific regions of the genome. Species-specific variations in the lengths of the fragments are analyzed by PCR amplification of specific DNA regions. The amplicons are then digested with restriction enzymes, and the lengths of the digested fragments are determined by gel electrophoresis, resulting in species-specific restriction profiles.

The profiles are compared with reference samples for species identification. This procedure has been widely used in seafood authentication because it is less costly, simpler, and more suitable for routine laboratory analysis than techniques such as FINS or DNA barcoding, both of which are based on DNA sequencing analysis. PCR-RFLP is a relatively rapid, reproducible, and robust laboratory technique that does not require expensive equipment, and it is approved in many countries for the determination of seafood species. PCR-RFLP is therefore well suited for fish species detection, particularly for use closer to the origin of the sample.

Researchers at Campden BRI in England have developed a PCR-RFLP method that replaces the gel electrophoresis step with microfluidic lab-on-a-chip technology, utilizing CE to analyze DNA fragments.2-3 Lab-on-a-chip CE increases the ease of use, sensitivity, speed, and reliability of PCR-RFLP compared to gel-based methods.1 The chips are single-use units that contain etched capillaries attached directly to sample loading wells (see Figure 1, above).

The five-step workflow for performing fish identification using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) on the Agilent Technologies lab-on-a-chip platform.
click for large version
The five-step workflow for performing fish identification using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) on the Agilent Technologies lab-on-a-chip platform.

The electrophoretic analysis and interpretation of results are completely automated, requiring only the click of a mouse after the samples are loaded onto the chip. The superior resolution of lab-on-a-chip technology enables the detection of DNA fragments that may be too small for visualization using gel electrophoresis. While the visual inspection of agarose gels and comparison to validated fish species patterns is tedious and error-prone, the lab-on-a-chip system automatically analyzes the pattern, compares it to a database of validated fish species patterns, and generates a species match. The database is expandable to thousands of species, assuring that this testing platform will be able to adapt to future needs.

How It Works

The lab-on-a-chip analysis starts with the extraction of DNA, which requires only 40 mg to one gm of tissue and is done using a solid phase extraction spin column (see Figure 2, p. 22). The second step is the amplification of the target DNA sequence by PCR, involving a 464 bp segment of the cytochrome b gene (cytb), a gene that is found in all vertebrate fish, with a sequence that is known in a large number of fish species. The amplified DNA fragment is then digested with three restriction enzymes (DdeI, HaeIII, NiaIII) to generate fragment size patterns specific to the fish species. A kit that provides all the required reagents for the amplification and restriction digests comes with the lab-on-a-chip system.

The fourth step involves loading the digested sample onto the chip and performing the automated electrophoretic separation of the restriction fragment. The final step is the automated analysis of the restriction fragment patterns by the onboard software that matches the patterns to a database of patterns from known species and identifies the most likely species (see Figure 3, below). The result is a fast and reliable species identification, even in samples from a mixture of fish species, with the ability to detect as little as 5% of a second species in the same sample.

Representative polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) patterns for nine fish species. They were identified automatically by the Agilent RFLP matcher software on the lab-on-a-chip platform using an onboard pattern database.
click for large version
Representative polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) patterns for nine fish species. They were identified automatically by the Agilent RFLP matcher software on the lab-on-a-chip platform using an onboard pattern database.

Several laboratories have already adopted the lab-on-a-chip PCR-RFLP platform for fish species identification. For example, Eurofins Scientific, an international group of laboratories that provides testing and support services to the food industry, has been using this technique for some time at their Wolverhampton, England, facility. The company has found the Agilent platform very useful, in part because the fragment-matching software was extremely helpful due to the large database of species available.

Having that much data available is key to helping solve a major problem. Seafood substitution is a significant component of a problem that, in total, costs the industry $10 to $15 billion per year, robbing it not only of revenues but also of the consumer confidence that is so vital to its health. Consumers’ chances of encountering fraud in the form of mislabeled fish are quite high every time they order fish in a restaurant or enter a fish market. While regulatory requirements and enforcement are part of the solution, the seafood industry recognizes the pressing need to eliminate fraud in all its forms.

Traditional methods of fish species determination have been tedious and often unreliable, but cutting edge technology in the form of lab-on-a-chip DNA testing is now available to provide fast, easy, automated, low-cost, and reliable results to meet the strong desire of industry and regulatory agencies to provide high-value products and maintain consumer confidence. ■

Zavitsanos is worldwide food business manager at Agilent Technologies Inc. Reach him at paul_zavitsanos@agilent.com.

References

  1. Rasmussen RS, Morissey MT. DNA-based methods for the identification of commercial fish and seafood species. Compr Rev Food Sci Food Safety. 2008;7(3):280-295.
  2. Dooley JJ, Sage HD, Brown HM, et al. Improved fish species identification by use of lab-on-a-chip technology. Food Control. 2005;16(7):601–607.
  3. Dooley JJ, Sage HD, Clarke ML, et al. Fish species identification using PCR-RFLP analysis and lab-on-a-chip capillary electrophoresis: application to detect white fish species in food products and an interlaboratory study. J Agric Food Chem. 2005;53(9):3348–3357.

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