Evaluation of locked nucleic acid and TaqMan probes for specific detection of cashew nut in processed food by real time PCR  Africa Sanchiz a , Isabel Ballesteros b , Eric Marqu  es c , M. Carmen Dieguez d , Julia Rueda c , Carmen Cuadrado a, *, 1 , Rosario Linacero c, 1 a Departamento de Tecnología de Alimentos, SGIT-INIA, Ctra. Coru~ na km 7.5, 28040 Madrid, Spain b Ingeniería en Biotecnología, Facultad de Ingenierías y Ciencias Aplicadas, Universidad de las Am ericas, Quito, Ecuador c Departamento de Gen etica, Fisiología y Microbiología, Facultad de Biología, Universidad Complutense de Madrid, 28040, Madrid, Spain d Servicio de Alergia, Instituto de Investigaci on Hospital 12 de Octubre (iþ12), Avenida de C ordoba s/n, 28041 Madrid, Spain article info Article history: Received 6 December 2017 Received in revised form 14 February 2018 Accepted 15 February 2018 Available online 16 February 2018 Keywords: Anacardium occidentale Allergen detection Real time PCR Moist thermal processing Processed foods abstract Cashew (Anacardium occidentale) nut can trigger serious reactions in allergic patients, including anaphylaxis and death. Labelling the presence of cashew nuts in food products is mandatory and consequently, sensitive and specific analytical methods must be developed. In this study, Ana o allergen coding sequences have been sequenced in several cashew varieties. Two hydrolysis probes, locked nucleic acid (LNA) and TaqMan, have been designed and their efficiency, sensitivity, limit of detection and specificity for Ana o 1 coding-sequence detection have been compared. Reliable Real Time PCR assays to detect and quantify up to 10 ppm of cashew nuts in complex mixtures have been developed. Moreover, the influence of boiling and autoclave treatment on cashew nut detectability has been analysed by qPCR, showing both probes similar performance. This analytical method was able to detect up to 1000 ppm with good functionality in autoclave treated samples. Boiling did not affect cashew nut detectability. Both hydrolysis probes are suitable for Ana o 1 coding sequence detection. Applicability of the assay has been studied by analysing several food products, and comparing the results with those of a commercial ELISA kit. © 2018 Elsevier Ltd. All rights reserved. 1. Introduction Tree nuts are valuable foods rich in proteins, minerals, vitamins, antioxidants and a considerable high content of unsaturated fatty acids (Ros, 2010), and their global production and consumption is increasing in the last years. Tree nuts allergy has also increased, although prevalence varies among individual nut and regions and it is difficult to stablish. In Europe, even though the most common allergies to tree nuts correspond to hazelnut and walnut, cashew nut allergy is getting importance over the last years according to several reports, with a variable prevalence among countries (Mendes, Costa, Vicente, Oliveira, & Mafra, 2016). Other studies indicate that allergies to walnut and cashew nuts are the most prevalent among tree nuts in the USA, achieving 20e30% and 15e30%, respectively (McWilliam et al., 2015). Originally from Brazil, cashew tree is cultivated in Africa and Asia, and mainly produced in India, and its fruits, cashews, are appreciated world- wide (Mendes et al., 2016). Cashew nuts are commonly consumed as snack or as ingredient in biscuits, sauces, sweets and several food dishes, and medical efficiency of cashew nuts has also been recently studied (Mah et al., 2017). Until the date, three major allergens have been identified, all of them being seed storage proteins (WHO-IUIS Allergen Nomenclature Sub-Comittee): Ana o 1, a 7S vicilin (Wang et al., 2002), Ana o 2, a 11S legumin (Wang, Robotham, Teuber, Sathe, & Roux, 2003), and Ana o 3, a 2S albumin (Robotham et al., 2005). In order to protect the safety of the allergic patients, European regulation obliges to advise the presence of tree nuts in food labels (Official Journal of the European Union (22/11/2011), 2011 Regula- tion (EU) No 1169/2011). The presence of allergenic ingredients in foods can be either a consequence of fraudulent substitution or adventitious contamination in the food facility. Last option is more likely for cashew nuts because of its high price. There is no treat- ment for food allergies, and sensitized individuals have to totally * Corresponding author. E-mail address: cuadrado@inia.es (C. Cuadrado). 1 Equal contribution. Contents lists available at ScienceDirect Food Control journal homepage: www.elsevier.com/locate/foodcont https://doi.org/10.1016/j.foodcont.2018.02.021 0956-7135/© 2018 Elsevier Ltd. All rights reserved. Food Control 89 (2018) 227e234