DNA-based Seafood Identification
Seafood is one of the most highly traded commodities in the world. In the interest of public health, it is vital that both domestically processed and imported seafood is safe, wholesome, and properly labeled. Under the Federal Food, Drug, and Cosmetic (FD&C) Act, the Fair Packaging and Labeling Act (FPLA) and the Public Health Service (PHS) Act, the Food and Drug Administration (FDA) facilitates programs that include inspection, sampling, analysis, research, and education on seafood issues. These programs assist the agency with its effort to oversee food safety and economic deception. The detection of species substitution is critical because it will assist in identifying and controlling species-specific hazards and reduce economic fraud.
There are numerous potential health risks associated with misbranding seafood species. For example, in 2007 several serious illnesses resulted from the illegal importation of toxic pufferfish that had been mislabeled as monkfish to circumvent U.S. import restrictions for this product (Cohen et al. 2009). Additional examples of species-specific hazards are listed in the FDA Seafood Hazard Guide: Food and Drug Administration Fish and Fisheries Products Hazards and Controls Guidance, Fourth Edition (2011)]. To aid in the proper labeling of seafood, the FDA maintains a list of acceptable market names for seafood sold in U.S. interstate commerce The Seafood List (FDA 2012).
One challenge faced by both consumers and regulators is the detection of "seafood substitution" in the marketplace - where a low value species or a species with a potential food safety hazard is mislabeled and substituted in whole or in part for a more expensive species or for a species with no potential food safety hazard. Substituted and/or mislabeled seafood is considered to be misbranded by the FDA and is a violation of Federal law.
It is not always possible to tell by simple inspection of an aquatic product that misbranding has occurred. Processing often removes or damages diagnostic characteristics crucial for the identification of species by conventional taxonomic means. Therefore, traditional morphological methods are often insufficient to provide for species resolution.
Several years ago, the FDA produced a web-based resource known as the Regulatory Fish Encyclopedia (RFE) to aid in the identification of commercially important species of fish (Tenge et al. 1997). Organized in a series of species "pages", the RFE contains high resolution images of whole fish and their marketed product forms (e.g. fillets, steaks), as well as other taxonomic, geographic, and relevant tools for species identification. An example of an identification method listed in the RFE is protein identification by isoelectric focusing (AOAC 1980). Isoelectric focusing is a currently accepted tool employed in the identification of fish fillets for regulatory compliance, but such analysis requires subjective interpretations of gel results and the inclusion of perishable frozen tissue standards in each run. Further, the technique is not effective in the case of processed or cooked samples. The RFE was designed so that it could be expanded to include additional data and to accommodate the use of newer analytical tools as they became available. In 2007, using methods developed as part of the Barcode of Life initiative, DNA barcode sequences were generated for 172 individual authenticated fish representing 72 species from 27 families contained in the RFE and the utility of DNA barcoding for regulatory seafood identifications was demonstrated (Yancy et al. 2008a).
The Barcode of Life initiative represents an ambitious effort to develop an identification system for eukaryotic life based upon the analysis of sequence diversity in short, standardized gene regions. Work is furthest advanced for members of the animal kingdom. In this case, a region of the cytochrome c oxidase subunit 1 gene (COI) has been targeted and pilot studies have shown its effectiveness in species identification. Even relatively short nucleotide sequences (100-200 bp) from the barcode region can provide accurate identifications in some cases, allowing barcodes to identify specimens whose DNA is degraded (Hajibabaei et al. 2006).
The Fish Barcode of Life campaign (FISH-BOL ) is a collaborative international research effort which seeks to establish a reference library of DNA barcodes for all fish species derived from voucher specimens with authoritative taxonomic identifications (Ward et al. 2009). Fishes comprise nearly half of all vertebrate species; the group includes approximately 15,700 marine and 13,700 freshwater species (FishBase ). Once completed, the FISH-BOL database will enable a fast, accurate, and cost-effective system for molecular identification of the world's icthyofauna. While the current FISH-BOL database contains a majority of sequences with proper authentication and traceability, this database is not currently searchable against only vouchered species with authoritative taxonomic identifications. There are plans to change this, but in the interim, FDA will only make regulatory decisions based on identifications using adequately authenticated standards.
Well-regimented, largely automated protocols for DNA barcode analysis have been developed at institutions such as The Canadian Centre for DNA Barcoding (CCDB) and the Smithsonian Institute's Laboratories for Analytical Biology (LAB). An example of a standardized fish barcoding analytical chain includes DNA extraction using a Glass-Fiber protocol (Ivanova et al. 2006) followed by PCR amplification using pre-made PCR plates (Hajibabaei et al. 2005, Ivanova & Grainger 2006) with primer cocktails that allow the amplification of barcode sequences from all fish species (Ivanova et al. 2007).
The FDA's Center for Food Safety and Applied Nutrition and Center for Veterinary Medicine, in collaboration with the University of Guelph's Biodiversity Institute of Ontario, in Canada, and the Laboratories of Analytical Biology at the Smithsonian National Museum of Natural History, Suitland, MD, USA, first provided details on protocols, reagents, and equipment required to carry out a validation study for barcode generation for fish identification in the form of FDA Laboratory Information Bulletin No. 4420 (Yancy et al. 2008b). In 2008, this protocol was evaluated by three laboratories in an inter-laboratory trial and the results of this trial were used to further refine the method. This modified version of the method was then subjected to a formal single laboratory validation (SLV) at CFSAN and published in the Journal of AOAC (Handy et al. 2011). A step by step protocol based on this published SLV study, with minor modifications, is provided below. For FDA purposes, this SOP is intended to replace LIB No. 4420.
- AOAC. (1980). Official Method 980.16 Identification of Fish Species: Thin-Layer Polyacrylamide Gel Isoelectric Focusing Method. J. AOAC 63:69 (1980); corr. 684.
- Cohen NJ, Deeds JR, Wong, ES, Hanner RH, Yancy HF, White KD, Thompson TM, Wahl M, Pham T, Guichard FM, Huh I, Austin C, Dizikes G, Gerber S. (2009) Public health response to puffer fish (tetrodotoxin) poisoning. Journal of Food Protection 72(4):810-817.
- FDA. (2011). Food and Drug Administration Fish and Fisheries Products Hazards and Control Guidance, Fourth Edition. (accessed 3/18/2013).
- FDA. (2012). The Seafood List: FDA Guide to Acceptable Market Names for Food Fish Sold in Interstate Commerce. Office of Food Safety, Division of Seafood Safety, FDA Center for Food Safety and Applied Nutrition, US Department of Health and Human Services. (accessed 3/18/2013)
- Hajibabaei M, Smith, A, Janzen, DH, Rodriguez, JJ, Whitfield, JB, Hebert, PD. (2006). A minimalist barcode can identify a specimen whose DNA is degraded. Molecular Ecology Notes 6:959-964.
- Handy, SM, Deeds, JR, Ivanova, NV, Hebert, PDN, Hanner, R, Ormos, A, Weigt, LA, Moore, M, Yancy, HF. (2011). A single laboratory validated method for the generation of DNA barcodes for the identification of fish for regulatory compliance. J. AOAC. 94(1):201-210.
- Ivanova N, Grainger C. (2006). Pre-made frozen PCR and sequencing plates. CCDB Advances (pdf), Methods Release No. 4. Biodiversity institute of Ontario, (accessed 7/16/2010).
- Ivanova NV, deWaard JR, Hebert PDN. (2006). An inexpensive, automation-friendly protocol for recovering high-quality DNA. Molecular Ecology Notes 6:998-1002.
- Ivanova NV, Zemlak TS, Hanner RH, Hebert PDN. (2007). Universal primer cocktails for fish DNA barcoding. Molecular Ecology Notes, 7:544-548.
- Tenge BJ, Dang NL, Fry FS, Savary WE, Rogers PL, Barnett JD, Hill WE, Wiskerchen JE, Wekell MM. (1997). Integration of Computer and Laboratory Techniques for Species Identification Including Development of a Regulatory Fish Encyclopedia. in Fish Inspection, Quality Control, and HACCP - A Global Focus, p. 214-226. Martin, Collette, and Slavin, (eds.), Technomic Publishing, Lancaster, PA.
- Ward RD, Hanner R, Hebert PDN. (2009) The Campaign to DNA Barcode all Fishes, FISH-BOL. Journal of Fish Biology 74:329-356.
- Yancy HF, Zemlak TS, Mason JA, Washington JD, Tenge BJ, Nguyen NT, Barnett JD, Savary WE, Hill WE, Moore MM, Fry FS, Randolph SC, Rogers PL and Hebert PDN. (2008a). The Potential Use of DNA Barcodes in Regulatory Science: Applications of the Regulatory Fish Encyclopedia. Journal of Food Protection 71(1):210-7.
- Yancy HF, Fry FS, Randolph SC, Deeds JR, Ivanova NV, Grainger CM, Hanner R, Weigt LA, Driskell A, Hunt J, Ormos A, Hebert PDN. (2008b) LIB No. 4420 A Protocol for Validation of DNA-Barcoding for the Species Identification of Fish for FDA Regulatory Compliance. FDA Laboratory Information Bulletin Vol. 24.
The procedure for establishing FDA Acceptable Market Names is described in Guidance for Industry: The Seafood List - FDA's Guide to Acceptable Market Names for Seafood Sold in Interstate Commerce.
FDA's guidance documents, including this guidance, do not establish legally enforceable responsibilities. Instead, guidances describe the Agency's current thinking on a topic and should be viewed only as recommendations, unless specific regulatory or statutory requirements are cited. The use of the word should in Agency guidances means that something is suggested or recommended, but not required.