Authentication of Canned Tuna and Bonito by Sequence and Restriction Site Analysis of Polymerase Chain Reaction Products of Mitochondrial DNA

Abstract

Methods of authenticating already canned fish were developed, using polymerase chain reaction (PCR) followed by sequencing and restriction site analysis. The canning process degrades DNA to fewer than 123 base pairs (bp) in length. Therefore, degenerate PCR primers were designed to amplify short (<123 bp) mitochondrial cytochrome b gene sequences known to differ at specific nucleotides among the species of interest. Sequences of canned tuna (Thunnus albacares, Thuunus alalunga, and Katsuwonus pelamis), bonito (Euthynnus affinis), and frigate mackerel (Auxis thazard) were reproducibly identified, and were used to determine which species or whether more than one species was present in individual cans. Restriction site analysis of two amplified regions of the cytochrome b gene demonstrated a faster and less expensive method than sequencing for distinguishing PCR products of different species. Thus, restriction site analysis of PCR products can be used in conjunction with sequencing to authenticate species in...