Fish species identification methods

In recent decades a number of polymerase chain reaction (PCR)-based methods for fish species identification by DNA analysis have been developed to support food control authorities to achieve this goal. In the following sections these techniques are described... 

Species identification of seafood product is important for the implementation of the labeling regulations as set by many countries. These regulations to prevent the substitution of some commercially important fish can be effectively achieved when species-specific data of all fish species are available. Genome and protein techniques are the two valuable methodologies that can be used for fish species identification. These methods can prevent adulteration of toxic puffer species as nontoxic puffer one. Hence, the development of genome and protein techniques for effective identification of puffer species is critically needed. 

C Pineiro, CG Sotelo, I Medina, JM Gallardo, RI Perez-Martin. Reversed-phase HPLC as a method for the identification of gadoid fish species. Z Lebensm Unters Forsch A 204 411-416, 1997. 

Molecular microbial ecology methods are being developed that fink species identification with activity, typically combining molecular identification methods with isotope labefing. For a comprehensive review of these methods, see Neufeld et al. (2007). One of these approaches combines isotope labeling followed by microautoradiography and hybridization techniques such as FISH (FISH-MAR) and microarrays (isotope arrays). FISH-MAR has been done with H labeled substrates (Cottrell and Kirchman, 2000 Lee et al., 1999 Ouvemey and Fuhrman, 1999), labeled substrates (Lee etal, 1999), and Pi labeled substrates (Lee ei estuarine water with H labeled amino acids, proteins. 

Digestion of the products with different endonucleases, followed by agarose gel electrophoresis of the digested products, yielded specific restriction patterns that enabled direct visual identification of the species analyzed. This PCR-RFLP methodology allowed not only clear discrimination of different salmon species in raw and smoked products but also of different fish species that may be present in food products. Russell et al. (2000) demonstrated that this method can be used to differentiate between salmon species. The reliability and practicality of the method was also tested by a collaborative study carried out in five European laboratories (Hold et al., 2004). 

The need for high-throughput screening methods of human mutations has stimulated the development of CE-based methods for SSCP analysis. For detection of ssDNA, PCR is carried out with primers labeled at the 5 site with fluorescence dyes. Two different labels may be used for identification of the forward and reverse strands. Advantages of CE-SSCP are speed of electrophoresis (ca. 10 min), high sensitivity, reproducibility, and the possibility of automation (Andersen et al., 2003 Hestekin et al., 2006). In food analysis, CE-SSCP has been used to identify bacteria (see Section 5.4.4) but, to the knowledge of the author, not to species identification of meat, fish, or other food up to now. 

Canning of fish is accompanied by severe degradation of DNA (Mackie, 1999). Recently, Chapela et al. (2007a) compared four different methods of extracting DNA from muscle of canned mna for PCR-based species identification. The smdy included amplification of sequences differing in length between 100 and 300 bp. Increased size... 

The same methods used for analysis of heat-treated products (see Section 11.2.2) could be employed for the species identification of fish meal (Rehbein, 2002). 

Five years ago a method to identify species solely by their DNA sequence was introduced under the term DNA barcoding (Hebert et al., 2003). A short fragment of a single gene (cytochrome c oxidase subunit I) has been selected for fish (Ward et al., 2005) and for many other vertebrate and invertebrate species. Up to now, sequences of more than 5100 fish species have been collected (www.fishbol.org), which can also be used for identification of commercially important fish species. It has to be expected that DNA barcoding will become a valuable tool to be used for control of traceability and labeling of seafood, as exemplified by some recent publications (Smith et al., 2008 Ward et al., 2008 Yancy et al., 2008). 

The detection systems used with HPLC can be broadly divided into three approaches photometry, plasma techniques (ICPAES, ICPMS), and cold vapour atomic absorption and fluorescence spectroscopy (CV-AAS, CV-AFS). The method with the lowest limits of detection (LOD) with sample introduction via a direct injection nebulizer used ICP-MS. An HPLC system coupled to atmospheric pressirre chemical ionization MS was used to identify methyl mercury spiked into a fish tissue CRM (DORM-1, NRCC). This type of system has a significant advantage over elemental detection methods because identification of the species present is based on their structure, rather than matching the analyte s retention time to that of a standard. 

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