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Development and validation of new methods for the qualitative detection and quantification of fish, crustacea and molluscs besides of insects as potential food allergens (AQUALLERG-ID)

04/2019-03/2022

This third-party funded project is conducted in the framework of the BfR research programme on authenticity testing of food and feed.

BMEL support programme: Promotion of innovation

BLE grant number: 281A103016

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Project description:

The project AQUALLERG-ID aims to close analytical gaps in terms of species rich allergen groups such as fish, crustacea and molluscs. For this purpose modern molecular biological methods as well as new quantitative ELISA (Enzyme-Linked Immunosorbent Assays) and qualitative immune chemistry based rapid tests will be developed to enable allergen control in place. To achieve this goal, the project partners will go different, but with view on the overarching aim, complementary ways. At BfR the parallel detection of more than fifty commercial fish and crustaceous species will be tackled by flexible low density PCR (polymerase chain reaction) arrays which will be pre-spotted on user friendly PCR plates. By using individual systems for different species groups/taxon level the problem of divergent sensitivities of hitherto existing universal detection systems will be overcome. The use of the method will be open to any laboratory after the project’s end. The company IMGM GmbH, utilizing modern Next Generation Sequencing (NGS), paves the way to a completely new multiple detection principle. NGS technology might revolutionize allergen analysis in the future. Finally at ifp new optimized immunological assays targeting aquatic species and fish products will be developed for the market. Apart from fish and crustaceous organism also molluscs are investigated, for which at present no commercial rapid tests are available by now. For the first time also insects as novel potentially allergenic food ingredient will be tackled. The project will be supported by the company FRoSTA AG by materials and consulting from the viewpoint of aquatic food industry.

BfR part of the project:

The aim of BfR’s project part is the development of a robust and reproducible screening-assay for the detection of species-specific DNA derived from aquatic organisms. For this, reliable real time PCR (polymerase chain reaction) methods will be employed in an efficient technical variation based on the principle of “Low Density-Arrays” using 384 micro-cavities. Hence substantially more species and/or groups of organisms can be tackled in one analytical step, circumventing frequently occuring specificity and sensitivity problems.

The work program comprises the development of new real time PCR systems as well as the adaptation of published systems. The approach will end in a “ready-to-use” format. The operator only has to apply extracted DNA and mastermix into the small-sized cavities (~ 3-10 µl) of pre-prepared PCR plates. All reagents (primer/probes) needed will have to be spotted and stabilized by drying. Plate spotting will be done autometically to achieve a high degree of reproducibility.

The assay encompasses species of regional meaning but also species which are rarely sold in Germany but do play an important role on the global markets and import. The choice of the species will be made on basis of a sound data base research. The selection of real time PCR systems, in addition, affords the adaptation to a unique temperature/time program. On the one hand, approaches will be developed using universal SybrGreen-Mastermixes without probes. This kind of screening decreases the costs for multiple analysis significantly. On the other hand, in parallel primer/probe-assays will be developed to meet the requirements of standard methods to include a confirmation reaction.

The assays shall be published and made available to any laboratory without restrictions. Furthermore, in close cooperation with the company IMGM GmbH (Martinsried, Bavaria, Germany), novel approaches will be developed based on NGS (Next Generation Sequencing) technology, enabling the multiple sequencing of species specific DNA by metabarcoding.

Project partners:

• Ifp, Germany
• IMGM Laboratories GmbH, Germany

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