Sesame, Pistachio, and Macadamia Nut: Development and Validation of New Allergenic Systems for Fast Real-Time PCR Application.

Food allergy is a worldwide health problem that concerns infants to adults. The main health risk for sensitised individuals is due to the presence of traces of allergens as the result of an accidental contamination during food processing. The labelling of allergens such as sesame, pistachio, and macadamia nut on food products is mandatory according to Regulation (EU) N. 1169/2011; therefore, the development of suitable and specific analytical methodologies is advisable. The aim of this study was to perform a multi-allergen real-time PCR system that works well in fast mode at the same annealing temperature and with the same thermal profile. The real-time PCR was developed designing new, specific, and efficient primer and probe systems for the 2S albumingene for sesame and pistachio and for the vicilin precursorgene for macadamia nut. These systems were subjected to a robust intra-laboratory qualitative validation process prior to their application, by DNA extraction and fast real-time PCR, on some real market samples to reproduce a potential allergen contamination along the food chain. The developed system results were specific and robust, with a sensible limit of detection (0.005% for sesame; 0.004% for pistachio; 0.006% for macadamia nut). The performance and the reliability of the target systems were confirmed on commercial food samples. This molecular approach could be used as a screening or as a support tool, in association with the other widespread monitoring techniques (such as ELISA).

New biomolecular tools for aerobiological monitoring: Identification of major allergenic Poaceae species through fast real-time PCR.

Grasses (Poaceae) are very common plants, which are widespread in all environments and urban areas. Despite their economical importance, they can represent a problem to humans due to their abundant production of allergenic pollen. Detailed information about the pollen season for these species is needed in order to plan adequate therapies and to warn allergic people about the risks they take in certain areas at certain moments. Moreover, precise identification of the causative species and their allergens is necessary when the patient is treated with allergen-specific immunotherapy. The intrafamily morphological similarity of grass pollen grains makes it impossible to distinguish which particular species is present in the atmosphere at a given moment. This study aimed at developing new biomolecular tools to analyze aerobiological samples and identifying major allergenic Poaceae taxa at subfamily or species level, exploiting fast real-time PCR. Protocols were tested for DNA extraction from pollen sampled with volumetric and gravimetric methods. A fragment of the matK plastidial gene was amplified and sequenced in Poaceae species known to have high allergological impact. Species- and subfamily-specific primer-probe systems were designed and tested in fast real-time PCRs to evaluate the presence of these taxa in aerobiological pollen samples. Species-specific systems were obtained for four of five studied species. A primer-probe set was also proposed for the detection of Pooideae (a grass subfamily that includes also major cereal grains) in aerobiological samples, as this subfamily includes species carrying both grass allergens from groups 1 and 5. These, among the 11 groups in which grass pollen allergens are classified, are considered responsible for the most frequent and severe symptoms.