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 JOHN J. DOOLEY,HELEN D. SAGE,MARIE-ANNE L. CLARKE, HELEN M. BROWN, AND STEPHEN D. GARRETT* Biochemistry Section, Department of Chemistry and Biochemistry, Campden & Chorleywood Food Research Association, Chipping Campden, Gloucestershire, GL55 6LD, UK Identification of 10 white fish species associated with U.K. food products was achieved using PCR- RFLP of the mitochondrial cytochrome b gene. Use of lab-on-a-chip capillary electrophoresis for end- point analysis enabled accurate sizing of DNA fragments and identification of fish species at a level of 5% (w/w) in a fish admixture. One restriction enzyme, DdeI, allowed discrimination of eight species. When combined with NlaIII and HaeIII, specific profiles for all 10 species were generated. The method was applied to a range of products and subjected to an interlaboratory study carried out by five U.K. food control laboratories. One hundred percent correct identification of single species samples and six of nine admixture samples was achieved by all laboratories. The results indicated that fish species identification could be carried out using a database of PCR-RFLP profiles without the need for reference materials. KEYWORDS: Capillary electrophoresis; food authenticity; species identification; PCR-RFLP; DNA fingerprinting; lab-on-a-chip; cytochrome b; fish species INTRODUCTION The diversity of fresh, frozen, and fish-based products available to the consumer has increased significantly in recent years. Products can range from premium-grade fish steaks to low-cost fish fingers. As fish are caught, processed, and distributed by a global network of operators, there is a need to ensure the authenticity and the origin of fish used in the products. This is especially true in the European Union (EU) where stringent fish catch quotas have been introduced in an attempt to limit the decline of native fish stocks. There is, therefore, a need to have reliable and simple species identifica- tion methods to support enforcement and compliance with labeling legislation (EC Council Regulation No. 104/2000 and EC Commission Regulation No. 2065/2001). Methods of fish species identification based on morphological characteristics are suited to whole or lightly processed fish; however, the identification of fish species becomes more difficult once it has been processed. The use of unique species- specific protein profiles has been reported for fish identification (1, 2); however, these methods are less reliable when applied to processed food products as the proteins become denatured. Furthermore, they require the analysis of species reference materials along with the samples. In terms of simplicity and speed, antibody-based methods would be most appropriate. However, only a limited number of immunoassays have been developed, and none are available for wide-scale commercial use (3, 4). DNA-based methods offer an alternative approach to species identification as DNA remains detectable in all but the most heavily processed samples. Direct sequencing is the most definitive method of identifica- tion; however, it cannot easily be applied to samples suspected or known to contain more than one species. Alternative techniques, using the polymerase chain reaction (PCR), have been applied to variable regions of DNA such as the cytochrome b or 5S rDNA genes. Although specific PCR assays have been used successfully to differentiate sole (Solea solea) and Green- land halibut (Reinhardtius hippoglossoides) species (5), in general more all-inclusive methods are used to identify a wider range of species. These methods include single strand confor- mation polymorphism (SSCP) (6), PCR-RFLP (restriction fragment length polymorphism) (7-12), and random amplified polymorphic DNA (RAPD) fingerprinting (13-15). A PCR-RFLP technique, which involves digesting an amplified 464 bp region of the cytochrome b gene with restriction enzymes to generate DNA profiles, has been used for the identification of a variety of species, including salmon, hake, sardine, eel, and flatfish (9, 11, 16; G. Hold, personal communication). Although useful for identification purposes, the PCR-RFLP technique relies on the use of gel electrophoresis and staining for endpoint detection, methods that are potentially hazardous * Corresponding author. Tel.: +44 (0)1386 842175; fax: +44 (0)1386 842100; e-mail: s.garrett@campden.co.uk. 3348 J. Agric. Food Chem. 2005, 53, 3348-3357 10.1021/jf047917s CCC: $30.25 © 2005 American Chemical Society Published on Web 03/31/2005