Analytical Methods Validating allergen coding genes (Cor a 1, Cor a 8, Cor a 14) as target sequences for hazelnut detection via Real-Time PCR Matteo D’Andrea, Jean Daniel Coïsson, Monica Locatelli, Cristiano Garino, Elisabetta Cereti, Marco Arlorio * Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche (DiSCAFF) and Drug and Food Biotechnology (DFB) Center, Università degli Studi del Piemonte Orientale ‘‘A. Avogadro”, via Bovio 6, 28100 Novara, Italy article info Article history: Received 17 December 2009 Received in revised form 22 June 2010 Accepted 11 July 2010 Keywords: Hazelnut Cor a 1 Cor a 8 Cor a 14 Real-Time PCR abstract Hazelnuts have been shown to contain different allergenic proteins. Amongst these, major allergens Cor a 1 and Cor a 8 and the 2S albumin Cor a 14 were selected as targets to comparatively validate three Real- Time PCR protocols. We investigated both on the choice of the amplification target, and on the matrix effect on different sample foods. Applying statistics on the validation parameters obtained from the three protocols, we showed a significant difference in Ct values. This could turn critical when a high sensitivity method is required for the detection of hazelnut traces, confirming how fundamental the choice of the template during primer design phase is. Concluding, statistical approach represents a useful tool for the identification of the best performing primer pairs in Real-Time PCR. Cor a 8 gene permitted the iden- tification of hazelnut based ingredients in complex foods, providing a significantly higher sensitivity in the PCR amplification, when compared to Cor a 1 and Cor a 14. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Food allergy is a crucial problem in the food safety manage- ment. Although there is uncertainty regarding the prevalence of food allergy, particularly to fruits, vegetables, nuts, and other edi- ble plants, recent extensive surveys showed how hazelnuts rank amongst the most important sources of food allergy in Europe, with a prevalence estimated to be between 0.1% and 4% (Zuidmeer et al., 2008), confirmed by double-blind placebo-controlled food challenges (DBPCFC) (Hansen et al., 2009). The allergen doses able to trigger objective symptoms in sensitised patients are difficult to calculate. Concerning the subjective response of sensitised pa- tients, the lowest dose inducing symptoms for hazelnut evaluated by DBPCFC was determined to be 1 mg of protein (Wensing et al., 2002). The only effective approach to treat food allergy is based on the total avoidance of allergen-containing foods in the diet. Nevertheless, sensitive individuals may be inadvertently exposed to allergenic proteins by consumption of food products supposed to be free of a certain allergen. Food products can be contaminated with foreign food constituents during all the possible food-process- ing steps (Poms, Anklam, & Kuhn, 2004). Hazelnut (Corylus avellana L.) seeds, belonging to widely con- sumed tree nuts, are used in a range of confectionery products (filled chocolates or wafers, cereal muesli mixtures, bakery prod- ucts as a basic component of nut fillings in various cakes, and oth- ers) (Piknová, Sangallo, & Kuchta, 2008). Hazelnuts have been shown to contain different allergenic proteins previously charac- terised (Pastorello et al., 2002). An 18 kDa major allergen was found to be related to Cor a 1, the major hazel pollen allergen with a high degree of homology with Bet v 1 (major birch pollen aller- gen). Cor a 8 is a lipid transfer protein (LTP) of 9 kDa; Cor a 9 is a tree pollen-unrelated hazelnut allergen. These protein families share little sequence homology, apart from the cysteine skeleton, but they have highly similar a-helical structures. This structure is highly stable and able to resist both to thermal and to proteolytic denaturation; this fact might contribute to their allergenicity (Radauer & Breiteneder, 2007). Recently, Cor a 14 (2S albumin) has been suggested as a novel putative minor allergen in hazelnut (Garino et al., 2010). The Directive 2006/142/EC European Community shows a list of potential allergenic ingredients to be mandatorily labelled on foods, which comprises cereals containing gluten, crustaceans, eggs, fish, peanuts, soybeans, milk and dairy products (including lactose), tree nuts, celery, mustard, sesame seeds, lupine, molluscs and sulphites of at least 10 mg/kg (Directive 2006/142/EC). Several analytical approaches were suggested to detect the presence of potential allergens in food products (Poms et al., 2004). ELISA ap- proach is currently considered the ‘‘gold standard” for the direct allergen detection. Several sandwich ELISA-based methods specific for hazelnut are nowadays commercialised (Garber & Perry, 2010). Also DNA-based methods (Polymerase Chain Reaction) are increas- ingly applied to the detection of foreign food ingredients, 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.07.029 * Corresponding author. Fax: +39 0321375751. E-mail address: marco.arlorio@pharm.unipmn.it (M. Arlorio). Food Chemistry 124 (2011) 1164–1171 Contents lists available at ScienceDirect Food Chemistry journal homepage: www.elsevier.com/locate/foodchem