| 英文摘要 |
Tuna is one of highly valuable seafood, and it is usually distributed as filleted, broiled and canned products. There are 8 species in Thunnus genus, and each species has very different commercial values, therefore the authentication of tuna species has become a very important issue. DNA based techniques have been extensively used recent years to validate and identify the species of tuna products. Real-Time PCR is one of bio-molecular approaches to identify the quality and quantity of target genes, and the process can be finished within 3 hours compared to traditional DNA sequencing method, which would take 3 days at least. In this research, our aim is to develop a rapid method for species identification by using probes and species specific primer to detect single-base variation of the target gene. We have successfully designed 2 SNPs (single nucleotide polymorphism) probes in mitochondrial cytochrome b gene, which can exclusively and specifically recognize T. macoyii and T.albacares, respectively. Moreover, we’ve developed two species specific primers based on SYBR Green method. One pair of the primer designed from ATPase gene can distinguish T. obesus from other species by analyzing Ct values. The other primers designed from D-loop gene can differentiate T. alalunga and T. orientalis from other species by Melting Curve Analysis method. These assays are useful application to rapidly discriminate phylogenetically close tuna species. |