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BIOTECON Diagnostics – A Life-threatening Risk

Allergens can be a deadly risk affected individuals cannot tolerate the upward tendency. There are a lot of triggering food consumption of certain . Their body reacts with components. Effective dose may a specific immune response to the uptake of particular be very low . The symptoms range from mild and Therapeutic approach reactions in the mouth, tongue and throat (swelling) as well In general, food allergy cannot be healed in most Proper allergen labeling as gastrointestinal symptoms (nausea, vomiting, ) patients. Although first success has been reported can save lives to respiratory symptoms (allergic ). The worst for hyposensitization therapies, this strategy is still case can be life-threatening anaphylactic shock requiring controversial. The only suitable approach for affected immediate medical treatment. patients is to avoid the allergen triggering foods. Thus, labeling of food products and reliable analytical methods are required. This has motivated BIOTECON Diagnostics GmbH to develope highly sensitive and specific molecular detection methods for more trusted food allergen labeling.

The Kiss of Death! In 2002, American physicians reported the case of a 20 year old woman with a known allergy to crustaceans. She nearly died due to allergic symptoms after kissing her boyfriend: Food worldwide in 2014, source: WAO. He had consumed some hours before! The kiss transferred traces of allergenic proteins and lead to an intense immune reaction. The symptoms were an Food allergy: a worldwide disease of the lips, a swelling of the throat, FLUSH-symptoms, urticaria, abdominal convulsion, respiratory filibuster and dyspnea. Food allergy is not a rarely occurring phenomenon: different Source: Steensma, DP: The kiss of death: a severe reaction to a sources estimate the prevalence of food allergy to be 2-4 induced by a good-night-kiss, Mayo Clinic Proceedings, 78(2002), % among grown-ups and 4-8 % among children with an P.221-222 Testing For Food Allergens – Legal Regulations

Different countries have Modern makes it difficult to identify if a implemented rules for particular product contains a relevant allergen. “Hidden” Special Case : labeling of food allergens allergens – allergenic food components which are not In contrast to “gluten-free” labeling of Codex Alimentarius, identifiable at first glance – are a risk for patients. For EU regulation clearly requires labeling of specified gluten- Risk of cross- example celery is widely used in spices, convenience containing cereals. This difference is based on the diseases which are the foundation contamination is treated products and ready-to-eat food. for the regulations. While the EU directive refers to allergy, Codex very differently Thus, different countries released regulations for the Alimentarius aims for affected persons. labeling of different allergenic compounds in food products. Threshold levels are often lacking

USA EUROPEAN UNION AUSTRALIA/NEW ZEALAND FALCPA (Food Allergen Labeling Regulation EU/1169/2011, VITAL (Voluntary Incidental Trace and Consumer Protection Act) Directives 2003/89/EU Allergen Labeling) System and 2007/68/EU

8 allergenic foods are 14 allergenic foods are 10 allergenic foods are subject to labeling subject to labeling subject to labeling Labeling if even cross contact to Only referring to ingredients, not Comprehensive support for allergenic substances is possible dealing with accidental cross- risk assessment and labeling contamination and traces of intentional and possible accidental allergens No thresholds for labeling No thresholds for labeling Threshold system for labeling Commercial system Which Allergens Must Be Tested?

Most foods have an allergenic potential The “Big Eight” Table 1. Food allergens - regional regulation and analytical approach Only a few food materials like , leaf lettuce or artichokes Allergen Subject to labeling in Detection by The majority of do practically never cause allergic reactions. More than 70 allergenic reactions different foods have been reported to trigger allergenic EU, CH, USA, CAN, AUS Immuno, PCR (and gluten containing cereals) are caused by a limited symptoms. number of food allergens However, the “Big Eight” are responsible for about 90% of EU, CH, USA, CAN, AUS Immuno, PCR

all allergenic reactions to food: Soya EU, CH, USA, CAN, AUS Immuno, PCR

Wheat, Peanut, Soya, , Milk EU, CH, USA, CAN, AUS Immuno Egg, Nuts, Crustacean, Fish Egg EU, CH, USA, CAN, AUS Immuno

Nuts (, , Extended allergen labeling in the EU , , , EU, CH, USA, CAN, AUS Immuno, PCR , and ) The EU defines 14 different food components, which are subject to labeling (see Table 1). Instead of only wheat, the Crustacean EU, CH, USA, CAN, AUS Immuno, PCR

EU requires labeling of gluten containing cereals, namely: Fish EU, CH, USA, CAN, AUS Immuno, PCR wheat, rye, barley, spelt, oats and kamut. Celery EU PCR

Mustard EU, CAN Immuno, PCR Additional information

It is important to mention that crustacean, fish and EU, CH, CAN, AUS Immuno, PCR molluscs are collective parameters: The many different species in these groups are not named separately and are not Lupin EU Immuno, PCR differentiated by the available testing methods. In contrast, nuts are clearly defined: almond, hazelnut, walnut, cashew, pecan Molluscs EU Immuno, PCR nut, brazil nut, pistachio and macadamia. There is no testing Chemical Sulfide EU, AUS method for detecting these as a collective parameter and single methods tests for each nut species must be used.

Abbrevations: European Union (EU), Switzerland (CH), United States of America (USA), Canada (CAN), Australia (AUS), Immunological based methods (Immuno) Allergen Testing – Why Real-time PCR?

Real-time PCR - Hydrolysis Probes

Food allergen analysis aims for the detection of the allergenic component in general since the pathologic potential of a sample is an individual value depending on Real-time PCR is ... 1. Denaturation (approx. 95°C) many factors and cannot be generally determined. ... fast, Benefits of testing by real-time PCR specific, DNA-based real-time PCR methods have been established sensitive and accepted in food analysis for many years now. They 2. Annealing ... (approx. 70°C 60°C) and safe! are used worldwide for the detection and quantification Primer

of bacteria, viruses, animal species, GMOs and allergens. Hydrolysis Probe Reporter Quencher Real-time PCR detects and amplifies a target DNA sequence using specific oligonucleotide primers and Table 2. Allergen detection methods - properties and limitations fluorescence probes. This technology enables a very ... and Elongation (approx. 60°C) PCR ELISA Lateral Flow specific, highly sensitive and – in combination with a Taq Polymerase

suitable reference material – even quantitative detection Detection of DNA Protein 40 cycles Light with defined of the target organism, including food allergens. Real- wavelength Medium - High signal detected time PCR is suitable to detect all allergens except milk Sensitivity High - very High High

and egg (low DNA content, PCR is not tissue specific) as Specificity Very High Medium - High Medium - High well as sulfide/sulfate (chemical parameter). Since DNA is no signal Quantitative Yes Yes No a very stable molecule, PCR can be applied also in highly

processed food matrices. Sample preparation is identical for Uniform sample prep for Yes No No all parameters all parameters. This enables time and cost saving workflows.

The detection of allergenic food components can easily be Automation for the entire Yes No No workflow from sample to integrated in existing PCR routines. result In contrast to protein-based allergen detection methods, Direct/indirect detection indirect Most cases Most cases after 40 cycles: real-time PCR is more specific, has a higher dynamic range of allergenic protein indirect indirect for quantification and delivers more consistent results. fluorescence intensity fluorescence cycles foodproof® Allergen Detection Kits

Available parameters: To protect customers effectively and ensure proper labeling, 7500 etc.). Using one DNA extraction, different allergens can Celery it is important to detect food allergens reliably, sensitively be determined within one PCR run due to harmonized PCR Soya and specifically. In context with modern food production, protocols. This saves time and costs for analysis. Hazelnut it is also important that testing times are short and assays The methods have been developed and validated based Peanut are stable and easy to handle. on our long experience in food testing. Walnut ® Gluten foodproof real-time PCR kits Allergen RM 800: BIOTECON Diagnostics has developed a series of real-time This innovative reference material contains all allergenic PCR kits to detect allergenic food components in all food food components in a non-allergenic matrix. The content matrices and environmental samples. Quantification is possible is adjusted precisely to 800 ppm. For quantification of food when used in combination with reference material. All assays allergens, this material can be extracted in parallel with the are easy to use and can be applied with all common real-time samples and measured in a dilution series. Therewith, the PCR devices able to detect 5’-Nuclease (TaqMan®) probes (e.g. allergen content of a food or raw material sample can be AriaMx®, LightCycler® 480, LightCycler® 96, PikoReal® 24, ABI reliably determined.

Technical Data:

64 reactions/kit Ready-to-use master mix including all reagents and Taq polymerase 5’Nuclease probes Detection of target (FAM-channel) and internal control (HEX-channel) Limit of detection: 0.1 ppm (referring to foodproof® Allergen RM 800) Limit of quantification: 0.8 ppm (referring to foodproof® Allergen RM 800) foodproof® Allergen Detection Kit foodproof® Sample Preparation Kits

Kits for manual and The basis for reliable detection of food allergens by real- easy to handle, needs little laboratory equipment and takes automated DNA time PCR is efficient DNA extraction. Since allergenic less than one hour. extraction are available food components are effective in low concentrations, it is necessary to extract and purify even small amounts of DNA Scale up your throughput High-throughput: from processed food and raw materials. For a high number of samples, the foodproof® Magnetic instruments for DNA Preparation Kit III in combination with a KingFisher Flex extraction and PCR foodproof® Sample Preparation Kits instrument offers a high-throughput method for DNA setup clearly increase As perfect addition to our real-time PCR kits, the foodproof® extraction used for food allergen detection. Up to 96 productivity technology comprises easy-to-use solutions for quick and samples can be processed in approximately 30 minutes. effective extraction of relevant DNA. Following cell lysis, the DNA is bound to the surface of The foodproof® Sample Preparation Kit III is a column based magnetic beads, which are automatically transferred to extraction system for manual application. After thermal and washing and elution buffers. This reduces manual effort chemical lysis, DNA is bound to the silica matrix of spin and speeds up analysis. filter tubes and purified in several washing steps before the A further improvement of productivity is achieved by clean DNA is dissolved in an elution buffer. This process is automated PCR setup using the foodproof® RoboSet+.

Manual DNA Extraction: Automated DNA Extraction: Automated PCR Setup:

foodproof® foodproof® KingFisher® Flex foodproof® RoboSet+ Sample Preparation Kit III Magnetic Preparation III Workflow Manual Manual

foodproof & Real-time microproof PCR Cyclers Detection Kits PRODUCTS & SERVICES

foodproof Automation DNA Extraction Kits Service Lab Automated Automated Analysis Results in < 2.5 h Sample Preparation DNA Extraction PCR Setup Real-time PCR Run Start approx. 30 min approx. 10 min 90 min

BIOTECON Detection and Quantification Kits Sample Preparation Kits Diagnostics GmbH A 500 13 Allergen RM 800 S 400 06.1 foodproof® Sample Preparation Kit III Hermannswerder 17 R 302 60 foodproof® Celery Detection Kit S 400 13 foodproof® Magnetic Preparation Kit III, 96 isolations 14473 Potsdam R 302 61 foodproof® Soya Detection Kit S 400 13 L foodproof® Magnetic Preparation Kit III, 480 isolations Germany R 302 62 foodproof® Hazelnut Detection Kit Phone: +49 (0) 331-2300-200 R 302 63 foodproof® Peanut Detection Kit Instruments Fax: +49 (0) 331-2300-299 R 302 64 foodproof® Gluten Detection Kit D 220 01 KingFisher® Flex [email protected] R 302 65 foodproof® Walnut Detection Kit Z 600 06 KingFisher® Flex Consumable Pack www.bc-diagnostics.com D 230 01 foodproof® RoboSet+ ® +

BR-ALLERGEN-EN V02/2016 D 230 01 HE foodproof RoboSet High-End Version