Materials of different food matrices were artificially contaminated (incurred) with defatted flours of peanut, hazelnut, cashew and walnut, respectively (hereafter referred to as “nuts”) and then processed. The composition of the materials used for in-house and inter-laboratory (= ring-trial) validation is compiled in Table 1. In this article, the design, intra- and inter-laboratory validation of a multiplex real-time PCR method “AllNut” for the simultaneous sensitive detection and semi-quantitative estimation of the allergenic foods peanut, hazelnut, cashew and walnut is presented. By the use of multicopy instead of singlecopy target sequences the sensitivity of the allergen detection can be increased significantly (Ladenburger 2018). The sensitivity (limit of detection) of these methods ranges between 5 and 20 mg/kg not covering such low concentrations as to protect very sensitive consumers. Several single and multiplex real-time PCR methods for detection of the tree nuts cashew, hazelnut and walnut have already been published (Iniesto et al. A real-time PCR method targeting a multicopy sequence from mitochondrial DNA has proven suitable for sensitive detection and even quantification of the allergenic ingredient peanut at the level of 5 mg/kg (Ladenburger et al. In routine analysis, predominantly protocols based on real-time polymerase chain reaction (PCR) and immunological methods have been used (EFSA 2014). The values are taking into account the published eliciting doses (Remington et al 2020). These action values serve as internal minimal thresholds by food control authorities and support laboratories in the decision-making process, whether further inspections at the production facility may be neglected or not (Waiblinger and Schulze 2018). The official food control laboratories in Germany are working with an internal action value of 1 mg protein/kg (derived from peanut or tree nut) or 5 mg/kg whole peanut or tree nut, respectively. An important factor for setting thresholds for food allergens is the availability of standardized methods for detection and quantification with sufficient sensitivity to detect even low amounts of allergenic components, if relevant. The implementation of legal threshold values is currently under discussion. In the case of positive findings, a quantification of the respective allergens or allergenic ingredients may be crucial for risk assessment, allergen management, and surveillance (The Allergen Bureau Waiblinger and Schulze 2018). Within the European Union, labelling is mandatory for food ingredients containing-amongst others-peanut, hazelnut, walnut and cashew (Regulation 2011/1169/EC). To ensure the availability of information about the presence of relevant allergens in food, labelling provisions have been set in many countries. However, the list of foods which account for most of the severe disease courses is rather short, namely peanut, tree nuts, fish, shellfish, egg, milk, wheat, soy, and seeds (Sicherer and Sampson 2018), with the first two being among the most frequent triggers for food allergies in Europe (Nwaru et al. The occurrence of food allergies can be observed more frequently among children than adults. There is extensive data suggesting that food allergies are already ubiquitous (up to 10% of people affected) with sharp increases mainly in the last 2–3 decades (Sicherer and Sampson 2018).
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