A Multiplex Real-Time PCR for the Sensitive Screening of Allergenic Foods, Including Mustard, Celery, and Pistachio

The objective of this study was to develop a sensitive, robust, and cost-effective real-time polymerase chain reaction (qPCR) system for the analysis of allergenic food ingredients, including white mustard (Sinapis alba), black or brown mustard (Brassica nigra, B. juncea), celery (Apium graveolens), and pistachio (Pistacia vera). The primary outcome of this research is to enhance the protection of allergy sufferers from undeclared allergenic ingredients or contaminants in food. The use of multicopy target sequences has demonstrated excellent sensitivity in the detection of various targets with detection limits of approximately 1 mg of the allergenic ingredient per kg (equivalent to less than 0.5 mg/kg of the respective protein per kg). The excellent sensitivity of the method was confirmed both intra- and interlaboratory by the analysis of model food matrices. The materials were incurred or spiked with allergenic food ingredients at concentrations ranging from approximately 1 to 20 mg/kg. The results of the specificity tests showed no cross-reactions with other plants. The evaluation of the results demonstrated the feasibility of quantifying the majority of the model food samples analyzed, despite the presence of very low levels of added allergenic ingredients. The method was deemed appropriate for the quantitative screening of allergens in mustard (white, brown, or black), celery, and pistachio, with a focus on detecting even very low traces of the respective allergens. A set of methods for allergen analysis is now available, comprising two other multiplex real-time PCR methods [16]. This set is both cost-effective and rapid, and valuable in terms of the number of allergens it covers. In total, it covers 10 different allergens. Furthermore, the mean values of the samples measured demonstrate that an estimation of the allergen concentration is possible, particularly if the source of the allergenic ingredient is known and can be utilized, for instance, for the calibration of the method. The results of the small-scale interlaboratory test show that the method is also suitable for a further collaborative trial.