Sequencing of Mitochondrial Cytochrome b Genes for the Identification of Meat Species

Abstract

DNA sequencing is a precise method for determining meat species. In order to amplify a target region from many species, we designed universal primers MI1 (5'-CAAATCCTCACA-GGCCTATTCCTAGC-3') and MI2 (5'-TAGGCGAATAGGAAATATCATTCGGGTTTGAT-3') from the published sequences of bovine, human, mouse and chicken mitochondrial genes. The PCR amplified 646bp fragments of mitochondrial cytochrome b genes from the 8 mammals (cow, sheep, goat, Japanese serow, sika deer, pig, horse, and rabbit) and the 5 birds (chicken, Japanese quail, Japanese tree-sparrow, dusky thrushe and a thrush in Europe). The sequences of the PCR products were determined via a direct method using a Dye Terminator Cycle Sequencing kit (ABI) and an ABI 373 A sequencer. The sequences of the 646bp fragments from the cow, sheep, goat, pig and chicken were in agreement with the published sequences except for small variations: the differences from the published sequences were 0, 7, 16, 2 and 3 nucleotides in the 646bp sequences of the cow, sheep, goat, pig and chicken, respectively. Closely related species of caprinae, Japanese serow, sheep and goat, were indentified by comparing the sequences because of 67-72 differences in the 646 nucleotides. Meat species can be determined via digestion of the PCR product with restriction enzymes; the 8 mammals were determined via a combination of Taq I, Alu I and Hae III, and the 5 birds via Taq I.