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

Abstract

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).

Detection of Parvalbumin Fish Allergen in Canned Tuna by Real-Time PCR Driven by Tuna Species and Can-Filling Medium

“…For instance, Sanchiz et al [ 34 ] reported that they used “Ara h 6” as a peanut allergen-coding region for the determination of peanut allergens that are impacted by different processing methods. Additionally, Toricelli et al [ 35 ] highlighted the achievements of the 2S albumin gene, performed by real-time PCR, for sesame, pistachio, and macadamia detection in commercial food samples. The allergen-encoding amplification results of the canned tuna groups are given in Figure 1 A.…”

Unbiased Immunization Strategy Yielding Specific Nanobodies against Macadamia Allergen of Vicilin-like Protein for Immunoassay Development

“…MiAMP2 vicilin-like protein accounts for almost 20% of the protein abundance in macadamia . Thus, it is frequently employed for the detection of macadamia residuals. , We therefore aimed to develop a sandwich ELISA detection strategy with the allergen-specific Nbs against vicilin-like proteins of macadamia. Thus, Nbs with His-tag only (as opposed to HA- and His-tag) were generated to serve as the antigen capturing Nbs.…”

“…Torricelli et al, designed primers for the vicilin precursor gene as the target to develop a fast real-time PCR. The practical LOD was determined as 0.006% . Ito et al also reported a PCR-based detection technique with a detection limit of 1 pg of macadamia DNA .…”

“…The practical LOD was determined as 0.006%. 9 Ito et al also reported a PCR-based detection technique with a detection limit of 1 pg of macadamia DNA. 10 However, the residual level of vicilin-like protein is difficult to estimate by PCR-based detection results, due to the dynamic distribution of the contaminants.…”

“…7,8 To date, various analytical methods have been explored and applied to detect allergens, i.e., DNA-based assays (e.g., realtime PCR) and immunoassays (e.g., enzyme-linked immunosorbent assay (ELISA)). 9,10 The immunoassays based on the interaction between specific antibodies and allergenic proteins have been developed and are routinely applied as a preferred detection method in the food industry. Traditional immunoassays based on monoclonal or polyclonal antibodies depend heavily on the quality of the purified allergenic proteins and of the antibodies, and failure to provide good-quality reagents will lead inevitably to a low testing efficacy.…”