Simultaneous quantification of the food allergens soy bean, celery, white mustard and brown mustard via combination of tetraplex real-time PCR and standard addition

Food allergies are a significant public health concern throughout the world with various symptoms that range from mild to severe or even life-threatening. As the only option for the allergenic consumer is the strict avoidance of the allergenic food, in the European Union the declaration of the most frequent food allergens on the labels of products is required. However, traces of food allergens that may be present in foods due to cross-contact are not included in this regulatory provision. Therefore, thresholds above which labeling is mandatory are being discussed. The surveillance of such thresholds requires specific, sensitive and quantitative methods. For this purpose, a tetraplex real-time PCR method was established to quantify simultaneously trace amounts of the four allergens soy bean, celery, white and brown mustard. The quantification is achieved by using the standard addition method. The approach was validated with DNA extracted from lysate mixtures of boiled sausage and the standard materials at concentration levels of 1, 5, 10, 20, 40, 100 and 400 mg/kg. The parameters recovery, repeatability and robustness were evaluated and the limits of quantification of soy bean (8.5 mg/kg), brown mustard (2.6 mg/kg), celery (3.7 mg/kg) and white mustard (36.8 mg/kg) were determined. The method was applied to commercial food products labeled with one or more of the analytes or with a “may contain traces of …” statement.