Identification of the species origin of highly processed meat products by mitochondrial DNA sequences.

Institut fiir Genbiologische Forschung, Ihnestrasse 63; 14195, Berlin, Germany The increasing tendency toward processing agricultural products at or near the site of origin requires methods to identify the species of plant or animal used for the respective product. One of the major forms of conservation and processing of animal meat, particularly fish meat, relies on extensive cooking and subsequent canning procedures. The identification of the species from such preparations by conventional methods presents several difficulties. Classic methods used to identify species origins of meat usually depend on the stability of soluble proteins. Species-specific protein patterns can be obtained by gel electrophoresis. Immunological methods may be applied whenever intact proteins can be isolated. These methods, however, fail with highly processed samples such as cooked and canned meat. A protocol for species identification of processed meat by sequence analysis of the mitochondrially encoded gene for cytochrome b has been published. ~1> This protocol was used successfully to amplify a 307-bp fragment of the cytochrome b gene and to identify the species origin of various slightly processed tissue samples, including canned salmon meat. Although not detailed in this report, canned salmon is usually not cooked and autoclaved as is canned tuna meat but, rather, is smoked and canned in plant oil without extensive heat treatment. In our hands, however, this method failed to identify the species origin of canned tuna meat, as no amplification was observed. To expand the range of this application to identify the sources of processed meat, we have investigated the potential of the cytochrome b gene for species identification of highly processed meat. Although more stable than the native configuration of proteins, DNA is also degraded by the action of radicals and heat. Progressive treatment and increased processing of meat products will thus increase the degradation of DNA and intensify the difficulties of recovering analyzable DNA. We now present an alternative procedure that allows the identification of tuna fish species even after extensive processing. In addition, we have introduced a cloning step before sequence analysis that enables us to analyze mixtares of more than one species. We chose the cytochrome b gene as a molecular marker for several reasons. First, because of the maternal inheritance of mitochondria, normally only one allele exists in an individual and thus no sequence ambiguities are to be expected from the presence of more than one allele. Second, the high abundance of mitochondrial DNA in total cellular nucleic acid preparations allows more effective PCR amplifications in comparison to nuclear-encoded, single-copy genes. Third, in vertebrates the mutation rate of mitochondrial genes is -10-fold higher compared to nuclear genes. Thus, point mutations accumulate quickly enough to allow (in most cases) the discrimination of even closely related species. Finally, cytochrome b gene sequences have been, and are, extensively used in investigations of phylogenetic relationships within vertebrates. As a result, sequence data of many vertebrates are already available for comparisons. We have explored the potential of using mitochondrial cytochrome b sequences for the identification of cooked and canned tuna fish species in commercial preparations. Here, we report the succesful identification of five different tuna species in a total of 30 commercially distributed cans.