Molecular methods for the differentiation of species used in production of cod-fish can detect commercial frauds Giuseppe Comi a , Lucilla Iacumin a , Kalliopi Rantsiou a , Carlo Cantoni b , Luca Cocolin a, * a Dipartimento Scienze degli Alimenti, Universit a degli studi di Udine, via Marangoni 97, 33100 Udine, Italy b Dipartimento di Scienze e Tecnologie Veterinarie per la Sicurezza degli Alimenti, Universit a degli studi di Milano, via Celoria 10, 20121 Milan, Italy Received 17 May 2003; received in revised form 6 November 2003; accepted 6 November 2003 Abstract A molecular approach was used to differentiate eight species commonly used in the production of cod-fish. Since visual iden- tification can only be applied easily on whole fish, we used the PCR method to obtain a short fragment of the cytochrome B (cytB) gene that was then analyzed by RFLP, SSCP and DGGE. While RFLP and SSCP resulted in differentiation of only some of the species tested, DGGE was able to produce patterns that made possible the identification of the species considered. The application of molecular methods to the identification of the species in this study was found to be useful, fast and reliable. Ó 2003 Elsevier Ltd. All rights reserved. Keywords: Identification; Cod-fish; PCR-based techniques; cytB gene 1. Introduction In the last few years, consumers have become more and more demanding in the choice of foodstuff. Important parameters that drive this selection are the safety and the correspondence of the ingredients with what has been declared on the label. This fact calls for the availability of reliable and rapid methods to assess the hygienic quality of food and to identify food com- ponents. Especially for meat or fish-based foods, meth- ods able to differentiate and identify species commonly used, must be developed. The necessity to identify dif- ferent species in foodstuff is an important aspect to consider when allergic problems towards specific species, or ethical/religious issues are taken into account. Moreover the definition of the species contained in a specific preparation is a crucial step in the food quality control to avoid possible commercial frauds. In this last case, it is very important to assess that species of high commercial value are not sold, partially or entirely substituted with other species of lower commercial value. An increasing number of studies and techniques have become available to identify different animal species in food. Histological analysis of tissues, fatty acids com- position, antigen–antibody gel diffusion (Kangethe, Gathuma, & Lindqvist, 1986), SDS-PAGE (Zerifi, Labie, & Bernard, 1991), ELISA-assays (Andrews, Berger, Mageau, Schwab, & Johnston, 1992; Martin, Wardale, Jones, Hernandez, & Patterson, 1991) and Isoelectricfocusing (IEF) (King, 1984; Renon, Colombo, Colombo, Biondi, & Malandra, 2001) are only some of the numerous techniques available for the species iden- tification. Recently, molecular methods based on nucleic acids amplification (PCR) have been developed and employed to reach the goal of the species differentiation. Usually PCR is coupled with other techniques able to detect differences in the sequence of the products obtained by PCR amplification. So far restriction fragment length polymorphism (RFLP) (Meyer, Hofelein, Luthy, & Candrian, 1995) and single strand conformation poly- morphism (SSCP) (Rehbein, Kress, & Schmidt, 1997) have been the techniques most frequently used for this purpose. Regarding fish-species identification, methods such as IEF (AOAC, 1998), liquid chromatography (Osman, * Corresponding author. Tel.: +39-0432-590-759; fax: +39-0432-590- 719. E-mail addresses: lscocolin@uniud.it, lscocolin@dsa.uniud.it (L. Cocolin). 0956-7135/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2003.11.003 Food Control 16 (2005) 37–42 www.elsevier.com/locate/foodcont