Application of Relative Quantification TaqMan Real-Time Polymerase Chain Reaction Technology for the Identification and Quantification of Thunnus alalunga and Thunnus albacares ITZIAR LOPEZ AND MIGUEL ANGEL PARDO* Department of Food Technology, AZTI-Fisheries and Food Technological Institute, Txatxarramendi ugartea z/g, E-48395 Sukarrieta (Bizkaia), Spain A novel one-step methodology based on real-time Polymerase Chain Reaction (PCR) technology has been developed for the identification of two of the most valuable tuna species. Nowadays, species identification of seafood products has a major concern due to the importing to Europe of new species from other countries. To achieve this aim, two specific TaqMan systems were devised to identify Thunnus alalunga and Thunnus albacares. Another system specific to Scombroidei species was devised as a consensus system. In addition, a relative quantification methodology was carried out to quantify T. alalunga and T. albacares in mixtures after the relative amount of the target was compared with the consensus. This relative quantification methodology does not require a known amount of standard, allowing the analysis of many more samples together and saving costs and time. The utilization of real-time PCR does not require sample handling, preventing contamination and resulting in much faster and higher throughput results. KEYWORDS: Species identification; real-time PCR; DNA; Thunnus albacares; Thunnus alalunga; canned tuna INTRODUCTION During the past years fish species identification has been an important concern in order to label correctly the seafood products. Moreover, an increase in the consumption of fish has facilitated the importation of new species from other countries. In the case of Spain, the commercialization of Scombroidei species from South American countries has currently increased considerably. The problem appears when these species are fraudulently labeled as the most valuable tuna species. Accord- ing to the European Union labeling legislation (EU Regulation 1536/92) products labeled as white tuna can include only Thunnus alalunga (albacore), whereas those products labeled as light tuna must contain Thunnus albacares (yellowfin). When the external morphological characteristics of the fish are removed due to filleting or processing such as canning, the only possibility to authenticate the food product is by means of using a molecular marker. There are two big groups of molecular markers that have been extensively used during recent years: proteins and deoxyribonucleic acid (DNA). Protein analysis techniques are based on its physicochemical differences (1), whereas DNA techniques consist in the detection of any nucleotide variation within sequence (2). Nonetheless, when the product to be authenticated is highly processed (like canned tuna), the adequacy of each marker is critical. The utilization of electrophoretic analyses of proteins extracted from canned samples is unsuitable because of the irreversible changes on water solubility during the thermal treatment (3). However, the DNA molecule appears to be much more stable to thermal treatment than others. In fact, the use of DNA as a molecular marker has shown itself to be the most powerful tool for species identification (4). According to DNA analysis of canned tuna, many works have described different techniques based on restriction site poly- morphic fragments (RFLP) (5-7), forensically informative nucleotide sequencing (FINS) (6, 8), and others (9-11) with the aim of identifying Scombroidei species. However, these techniques are not able to detect reliably a specific species in a mixture, which is widespread in canning. Real-time Polymerase Chain Reaction (RT-PCR) technology is based on the detection and quantification by a high-quality optical detection instrument of a fluorescence reporter included within a specific fluorogenic probe. This methodology is routinely used for the quantification of genetically modified organisms (GMOs) in food (12). Moreover, recently RT-PCR has also been used for the identification of species such as beef (13), peanut (14), and gadoids (4, 15) and even for the semiquantification of beef in food (13). The main aim of this study was to achieve two relative quantification procedures to identify and quantify two of the most valuable commercial Scombroidei species from the canning industry. To our knowledge, this system is the first method capable of detecting and quantifying tuna species by RT-PCR. * Corresponding author (telephone +34-94-602 99 00; fax +34-94-687 00 06; e-mail mpardo@suk.azti.es). 4554 J. Agric. Food Chem. 2005, 53, 4554-4560 10.1021/jf0500841 CCC: $30.25 © 2005 American Chemical Society Published on Web 04/30/2005