Research On The Molecular Identification Methods Of Oceanic Tuna And Salmon Species

Tunas and salmons have high nutritions, and in recent years become economically important fishery worldwide. Because they are widely used for commercial processed production, closely related fish are often adulterated to highly priced species. For example, Thunnus tonggol, Auxis thazard, A. rochei, Sarda orientalis, Euthynnus affinis, Tetrapturus audax are often as the replacements for the highly priced tunas. And Esox reicherti, Ilisha elongata are the substitutes to highvalue salmons. As different kinds of species have extremely different nutritional values, allergy substances and greatly various prices, the food safety becomes the focus topic. While many countries develop their own legislation concerning foods and specifically food labelling, the judgement and implementation must be based on the rapid and accurate identification. For effective fishery management, especially ending the food frauds, and protection of consumers?rights, it is important to develop a molecular method for rapid and reliable identification of commercial tuna and salmon products.Here, the traditional qualitative PCR and duplex quantitative real-time PCR(qPCR) technology were used for the species identification in tuna and salmon samples, using closely related specimens as the negative control group. The 16 S rDNA and control region(CR) of mitochondrial DNA were selected as the reference genes markers for tuna and salmon species identification of duplex quantitative real-time PCR. And CR fragments of salmon species were amplified by loop-mediated isothermal amplification(LAMP), which was firstly applied to detect the fish species.Firstly, in established duplex quantitative real-time PCR system, the 16 S rDNA and control region(CR) of mitochondrial DNA were selected as the genes markers for genus Thunnus and five tuna species(T. maccoyii, T. obesus, T. albacares, T. alalunga, and Katsuwonus pelamis) identification, respectively. The genus Thunnus and five tuna species were the tested groups, and the closely related species, such as T. tonggol, A. thazard, A. rochei, S. orientalis, E. affinis, T. audax, were the negative controls. Subsequently, these target samples were successfully tested by duplex quantitative real-time PCR system. The established duplex quantitative real-time PCR system was furhter confirmed by the traditional qualitative PCR and clone sequencing. Secondly, the duplex quantitative real-time PCR technology was applied to salmon species identification in Chinese market. According the reports analyzation of genetical salmon information, the 16 S rDNA and control region(CR) of mitochondrial DNA were selected as the feature genes and designed to corresponding primers and probes for famliy Salmonidae and four salmon species(Salmo salar, Oncorhynchus keta, O. mykiss, O. nerka) identification, respectively. The established duplex qPCR system for species identification of target salmon samples had good consistency and repeatability, verified by the traditional PCR. The two different genes were simultaneously amplified by duplex qPCR system to improve convenience in detection, providing the technical support for tuna and salmon food in Chinese market.Finally, the LAMP method was firstly applied to salmon species identification. The CR gene was analyzed as the feature gene of salmon and fragment of CR was amplified from S. salar by LAMP primers. Thus, we established the salmon LAMP identification system, in which S. salar was as the tested species and the O. mykiss, O. keta, O. nerka species were used as the negtive control groups. The S. salar species was successfully identified that was consistently detected by duplex qPCR. While the LAMP displays the rapid and simple operation, our results indicated that the application of LAMP system for salmon species identification was limited by its worse repeatability and high false positive rate.