Ring trial validation of single and multiplex real-time PCR methods for the detection and quantification of the allergenic food ingredients sesame,almond, lupine and Brazil nut

The inter-laboratory (=ring-trial) validation of 4 food allergen quantification methods using real-time PCR is described. Three single real-time PCR methods for the specific detection and quantification of sesame, almond and Brazil nut were used. Additionally, a multiplex real-time PCR method combining the detection of sesame, almond, Brazil nut and lupine was tested in parallel. Matrix based calibrants (rice cookies) spiked (=incurred) with defined amounts of sesame, almond, lupine and Brazil nut were applied for quantitative evaluation. Cookies based upon wheat and rice flour as well as sauce hollandaise powder each incurred with these allergenic ingredients in the range of 10–123 milligram per kilogram were used as ring-trial samples. The lowest spike level of 10 mg/kg could reproducibly be detected by 6 of 7 PCR systems. In quantitative evaluation of the results, reproducibility standard deviations of approximately 50 % and below were obtained. In addition, the effect of the food matrix on allergen quantification was examined. The range of “recoveries” over all matrices and methods was from 43 to 109 %.     

Application of real-time PCR for tree nut allergen detection in processed foods

Abstract

Currently, food allergies are an important health concern worldwide. The presence of undeclared allergenic ingredients or the presence of traces of allergens due to accidental contamination during food processing poses a great health risk to sensitized individuals. Therefore, reliable analytical methods are required to detect and identify allergenic ingredients in food products. Real-time PCR allowed a specific and accurate amplification of allergen sequences. Some processing methods could induce the fragmentation and/or degradation of genomic DNA and some studies have been performed to analyze the effect of processing on the detection of different targets, as thermal treatment, with and without applying pressure. In this review, we give an updated overview of the applications of real-time PCR for the detection of allergens of tree nut in processed food products. The different variables that contribute to the performance of PCR methodology for allergen detection are also review and discussed.     

Cleaning and other control and validation strategies to prevent allergen cross-contact in food-processing operations

Food allergies affect an estimated 10 to 12 million people in the United States. Some of these individuals can develop life-threatening allergic reactions when exposed to allergenic proteins. At present, the only successful method to manage food allergies is to avoid foods containing allergens. Consumers with food allergies rely on food labels to disclose the presence of allergenic ingredients. However, undeclared allergens can be inadvertently introduced into a food via cross-contact during manufacturing. Although allergen removal through cleaning of shared equipment or processing lines has been identified as one of the critical points for effective allergen control, there is little published information on the effectiveness of cleaning procedures for removing allergenic materials from processing equipment. There also is no consensus on how to validate or verify the efficacy of cleaning procedures. The objectives of this review were (i) to study the incidence and cause of allergen cross-contact, (ii) to assess the science upon which the cleaning of food contact surfaces is based, (iii) to identify best practices for cleaning allergenic foods from food contact surfaces in wet and dry manufacturing environments, and (iv) to present best practices for validating and verifying the efficacy of allergen cleaning protocols.     

Simultaneous detection of allergenic fish,cephalopods and shellfish in food by multiplex ligation-dependent probe amplification

People suffering from food allergy rely on correct food labelling as the ingestion of minimal amounts of the respective allergen can trigger severe allergenic reactions. Probes for the detection of DNA from allergenic fish, shellfish and cephalopod species in food using multiplex ligation-dependent probe amplification were developed. The specificity and the sensitivity of the detection system were investigated. The limit of detection was 20 mg kg^?1 for scallop, fish and bivalve species and 100 mg kg^?1 for cephalopod, gastropod and crustacean species using self-prepared sushi spiked with the analytes in different concentration levels. The analysis of 10 commercial food samples demonstrates the applicability of the developed method and its suitability for food quality control. Therefore, the method can be used to monitor the compliance with labelling rules regarding food allergens.     

MoniQA (Monitoring and Quality Assurance)—an EU-funded Network of Excellence (NoE) Contributing Toward a Harmonized Approach to Food Safety Management and Method Validation—Including Food Allergens

Reliable detection and quantification of allergens are essential in order to protect allergic consumers and to comply with labeling regulations. In recent years various allergen-detection methods have been published, and test kits have become commercially available. Due to the nature of the analytes (usually allergenic proteins, specific marker proteins, or specific DNA markers) and their susceptibility to various processing effects, reliability and comparability of results have posed a great challenge. Often processing and matrix effects hamper the extraction efficiency and the quantitative analysis of allergens or markers in food products. Both reference methods and reference materials are urgently needed in the field of allergen testing. The EU-funded Network of Excellence, MoniQA—Monitoring and Quality Assurance in the Total food Supply Chain ()—is working toward the harmonization of monitoring and control strategies for food quality and safety assessment and thus focuses on performance criteria for methods used to analyze foods and food products for safety and quality. MoniQA established various analyte-specific working groups: Microbiological Contaminants, Mycotoxins and Phycotoxins, Chemical Contaminants, Food Allergens, Food Additives and Processing Toxicants, Food Authenticity, and Emerging Issues. MoniQA’s Food Allergen Working Group (WG) is compiling information about the most important food allergens, identifying gaps, prioritizing requirements, and developing harmonization guidelines in collaboration with all stakeholder groups, which include industry, food authorities, consumers, and laboratories. The WG works on (1) harmonized validation protocols and certification criteria for allergen testing, (2) status recognition of allergen methods which underwent a validation trial, (3) reference/testing materials, (4) international ring trials for full validation of new reference/testing materials and analytical methods, and (5) the development of a reference method by supporting research toward the improved use of mass spectrometry in food allergen testing. Additionally training for research and industry in the areas of analytical method development, method validation and verification, allergen management, and risk communication and a database on available analytical methods, validation level, and legislation linked with the RASFF—EU’s Rapid Alert System for Food and Feed are provided.     

A critical review of the specifications and performance of antibody and DNA-based methods for detection and quantification of allergens in foods

Despite the availability of a large number of antibody and DNA based methods for detection and quantification of allergens in food there remain significant difficulties in selecting the optimum technique to employ. Published methods from research groups mostly contain sufficient detail concerning target antigen, calibration procedures and method performance to allow replication by others. However, routine allergen testing by the food industry relies upon commercialised test kits and frequently the suppliers provide disappointingly little specification detail on the grounds that this is proprietary information. In this review we have made a critical assessment of the published literature describing the performance of both commercial and non-commercial test kits for food allergens over the period 2008–2018. Mass spectrometric methods, which have the potential to become reference methods for allergens, are not covered in this review. Available information on the specifications of commercial ELISA and LFD test kits are tabulated for milk, egg and peanut allergens, where possible linking to publications concerning collaborative studies and proficiency testing. For a number of commercial PCR test kits, specifications provided by manufacturers for detection of a small selection of allergen are tabulated. In conclusion we support the views of others of the critical need for allergen reference materials as the way forward to improve the comparability of different testing strategies in foods.     

Development of a Biological Assay to Determine the Allergenic Potential of Foods

Food allergies represent a risk for many people in industrialized countries. Unrecognizable allergenic proteins of foodstuffs may be present as ingredients that are not labeled or as unknown cross-contamination. Such hidden allergens can cause severe reactions in allergics, even at minute quantities, sometimes with fatal outcome. For the verification of the presence of allergenic food constituents, analytical methods such as ELISA and PCR have been developed. However, these tests cannot measure allergenic potential. For this reason, a test system that measures the biological activity of allergens has been developed. It is based on the cellular mechanisms of the type I allergy. Rat basophilic leukemia cells (RBL-2H3) were transfected with the genes of the human high affinity receptor for IgE. The resulting cell line expressed the human receptor α-chain and could bind allergenspecific IgE from allergic subjects, in contrast to the parent cell line. After cross-linking of receptor-bound, allergen-specific human IgE by allergens, the cells released measurable inflammatory mediators. These cells were used for the analysis of a variety of allergen extracts, including extracts prepared from foods containing allergenic hazelnut and peanut. The comparative validation with existing ELISA and PCR for hazelnut and peanut demonstrated similar sensitivity and specificity. The established cell line will be a novel tool in the detection of allergens in complex mixtures, especially to address the issue of their allergenic potential, which cannot be accomplished by classical analytical methods. This will add valuable information about the allergenic potential of food constituents to the risk assessment of foods.     

First ring-trial validation of real-time PCR methods for the quantification of allergenic food ingredients

The first interlaboratory validation of two food allergen quantification methods using real-time PCR is described. Methods for the specific detection and quantification of soybean and white mustard in boiled sausages were used. Matrix-based calibrants spiked with defined amounts of soybean and white mustard were applied for quantitative evaluation. The lowest spike level of 10?mg soybean and white mustard per kilogram could reproducibly be detected. Recovery in spiked sausages was between 82 and 99?% for soy and between 80 and 93?% for mustard. Reproducibility standard deviation was in the range that would be acceptable, for example, for quantitative GMO analytical methods (<35?%).     

Methods for allergen analysis in food: a review

Food allergies represent an important health problem in industrialized countries. Undeclared allergens as contaminants in food products pose a major risk for sensitized persons. A proposal to amend the European Food Labelling Directive requires that all ingredients intentionally added to food products will have to be included on the label. Reliable detection and quantification methods for food allergens are necessary to ensure compliance with food labelling and to improve consumer protection. Methods available so far are based on protein or DNA detection. This review presents an up-to-date picture of the characteristics of the major food allergens and collects published methods for the determination of food allergens or the presence of potentially allergenic constituents in food products. A summary of the current availability of commercial allergen detection kits is given. One part of the paper describes various methods that have been generally employed in the detection of allergens in food; their advantages and drawbacks are discussed in brief. The main part of this review, however, focuses on specific food allergens and appropriate methods for their detection in food products. Special emphasis is given to allergenic foods explicitly mentioned in the Amendment to the European Food Labelling Directive that pose a potential risk for allergic individuals, namely celery, cereals containing gluten (including wheat, rye and barley) crustaceans, eggs, fish, peanuts, soybeans, milk and dairy products, mustard, tree-nuts, sesame seeds, and sulphite at concentrations of at least 10 mg kg^-1. Sulphites, however, are not discussed.     

食品过敏原生物传感检测技术研究进展

食品过敏原已成为全球性的食品安全隐患。应对食品过敏反应最直接、有效的方法就是避免食用和接触各种含有致敏成分的食品。因此,快速和灵敏地检测食品中过敏原对预防和控制食品过敏反应是十分重要的。食品过敏原检测主要可以分为基于核酸和蛋白质的两大类检测技术。与基于聚合酶链式反应技术、液相色谱-串联质谱法的传统检测手段相比,生物传感技术检测平台因具有操作简单、高通量、灵敏度高、现场便携等优点,在食品过敏原快速检测领域备受关注。本文综述了近年来食品过敏原的生物传感检测技术研究进展,包括电化学传感器、光学传感器和其他新型传感器及其在食品过敏原检测中的应用,并展望了生物传感技术在食品过敏原分析中面临的挑战和发展趋势。     

食物过敏原标准物质的研究进展

食物过敏原标准物质是解决食物过敏问题的关键实验材料之一。在食物过敏原标准化方面,鸡蛋、牛奶和花生过敏原的分离纯化、结构和免疫学性质表征方面取得了良好的效果,为研发单个过敏原蛋白标准物质提供了支撑。在美国国家标准与技术研究院(NIST)颁布的8种过敏性食物标准物质中,只有RM8445用于检测食物过敏原;由欧盟资助的两大项目中,EuroPrevall项目主要构建了一个食物过敏原信息的数据库,而CREATE项目则首次证实重组花粉过敏原rBetv1可作为天然花粉过敏原Betv1的候选标准物质,为开发重组食物过敏原标准物质提供了一种可借鉴的策略。总之,研发食物过敏原致敏性蛋白标准物质是一项重要的任务,极具挑战性。      

食品中杏仁过敏原成分的检测

目的：建立检测食品中桃仁、杏仁过敏原成分的荧光PCR 方法,比较国外3 种ELISA 试剂盒效果。方法：针对杏仁Pru du1 基因设计引物及探针,建立荧光PCR 方法。利用杏仁过敏原参考物质对3 个品牌的ELISA试剂盒的回收率进行比较。结果：建立的荧光PCR 方法,具有很好的特异性;灵敏度为10mg/kg。结论：桃仁及杏仁过敏原成分荧光PCR 检测方法特异性好、灵敏度高,对食品中过敏原的检测有重要的实际意义。     

Advanced DNA- and Protein-based Methods for the Detection and Investigation of Food Allergens

Currently, food allergies are an important health concern worldwide. The presence of undeclared allergenic ingredients or the presence of traces of allergens due to contamination during food processing poses a great health risk to sensitized individuals. Therefore, reliable analytical methods are required to detect and identify allergenic ingredients in food products. The present review addresses the recent developments regarding the application of DNA- and protein-based methods for the detection of allergenic ingredients in foods. The fitness-for-purpose of reviewed methodology will be discussed, and future trends will be highlighted. Special attention will be given to the evaluation of the potential of newly developed and promising technologies that can improve the detection and identification of allergenic ingredients in foods, such as the use of biosensors and/or nanomaterials to improve detection limits, specificity, ease of use, or to reduce the time of analysis. Such rapid food allergen test methods are required to facilitate the reliable detection of allergenic ingredients by control laboratories, to give the food industry the means to easily determine whether its product has been subjected to cross-contamination and, simultaneously, to identify how and when this cross-contamination occurred.     

Quantification of lupine (Lupinus angustifolius) in wheat flour using real-time PCR and an internal standard material

In the European Union, labelling of the 14 food allergens listed in Annex IIIa of Directive 2000/13/EC is mandatory. The implementation of upper limits for these allergens is under discussion. Therefore, quantitative analytical methods will be needed to verify compliance with regulatory requirements and to provide an improved basis for the legal assessment of allergen labelling. In this study, the lupine flour content in wheat flours was determined using real-time PCR and statice seeds as internal standard material. The method proved to be applicable to the quantification of lupine contents from 1 to 10?mg/kg, which is in the range relevant for allergic consumers.     

Development and validation of a real-time PCR detection method for celery in food

As from 25 November 2005 onwards, a list of ingredients with known allergenic potential has to be labeled according to Directive 2003/89/EC, including celery and products thereof. In order to provide appropriate detection methods a novel real-time polymerase chain reaction (PCR) system for the specific and sensitive detection of DNA from celery (Apium graveolens) was developed and validated. Specificity was confirmed by testing DNA derived from more than 50 food relevant organisms. Sensitivity was demonstrated on the basis of a calibration curve plotting the corresponding Ct-values against DNA amounts ranging from 1 to 1000 copies. Due to the lack of certified reference material the applicability of the method was assessed by analysis of sausages spiked with defined amounts of grounded celery seed. The limit of detection (LOD) examined exemplarily for emulsion-type sausages was 5–10 mg/kg. Analysis of celery-containing commercial products demonstrated the performance potential and limitations of the new real-time PCR system.     

基于蛋白质和DNA的食品过敏原检测技术的研究进展

目前,食品过敏问题已成为全球性的健康问题。在食品加工过程中产生的食品过敏成分或微量过敏原对敏感机体都是巨大的健康威胁。因此,可靠的分析方法是鉴别和检测食品中过敏成分所必需的。该文综述了基于蛋白质和脱氧核糖核酸(DNA)的食品过敏原检测技术的应用及发展,并展望了其未来发展趋势。     

Food Allergens: Is There a Correlation between Stability to Digestion and Allergenicity?

Food allergy is a major health problem in the Western countries, affecting 3–8% of the population. It has not yet been established what makes a dietary protein a food allergen. Several characteristics have been proposed to be shared by food allergens. One of these is resistance to digestion. This paper reviews data from digestibility studies on purified food allergens and evaluates the predictive value of digestibility tests on the allergenic potential. We point out that food allergens do not necessarily resist digestion. We discuss how the choice of in vitro digestibility assay condition and the method used for detection of residual intact protein as well as fragments hereof may greatly influence the outcome as well as the interpretation of results. The finding that digests from food allergens may retain allergenicity, stresses the importance of using immunological assays for evaluating the allergenic potential of food allergen digestion products. Studies assessing the allergenicity of digestion products, by either IgE-binding, elicitation or sensitizing capacity, shows that digestion may abolish, decrease, have no effect, or even increase the allergenicity of food allergens. Therefore, the predictive value of the pepsin resistance test for assessing the allergenic potential of novel proteins can be questioned.     

Detection of celery ( Apium graveolens ), mustard ( Sinapis alba , Brassica juncea , Brassica nigra ) and sesame ( Sesamum indicum ) in food by real-time PCR

Legislation requires labelling of foods containing allergic ingredients, amongst them celery, mustard and sesame. Here we present robust quantitative and sensitive methods for real-time PCR detection of celery, mustard (Sinapis alba and Brassica sp.) and sesame in food. The development of the DNA-based assays was part of an effort to generate alternative detection methods for allergens for which effective protein-based assays are lacking. The celery and sesame methods were specific for the celery mannitol dehydrogenase gene and the sesame allergen encoding 2S albumin gene, respectively, when tested against a range of plant materials. The mustard method was specific for the allergen encoding sinA gene and its homologues present in different Brassica sp. All primer probe pairs gave high amplification efficiency and sensitivities below approximately ten molecules of purified template DNA. These DNA-based detection methods will constitute supplementary and complementary methods to the traditional protein-based methods. Laboratories may choose different analysis formats depending on the food matrix, the availability of specific tests and the performance characteristics of the tests.     

蜂花粉过敏原识别及微生物发酵法降解过敏原的潜在应用研究进展

蜂花粉是蜜蜂从显花植物上采集的花粉粒,并向其中加入花蜜及唾液腺分泌物而形成的团状物。蜂花粉营养物质和活性成分十分丰富,具有极高的药用价值。然而,部分易敏人群因食用蜂花粉产生临床过敏症状,限制了蜂花粉食用安全。然而目前国内外关于蜂花粉的致敏机制研究尚不完善,制约了蜂花粉进一步开发利用。本文通过对近年来国内外关于蜂花粉致敏性及其过敏原识别的相关研究现状进行综述,并对微生物发酵法降解过敏原的潜在应用进行分析讨论,以期为后续开发高效的蜂花粉脱敏技术提供参考。     

质谱技术在食物过敏原检测中的研究进展

食物过敏是当前食品安全领域比较突出的问题,相应的过敏原检测方法研究也越来越受重视。相比于传统的免疫学检测方法,质谱技术在检测加工后以及复杂基质中的食物过敏原中,具有高通量和高灵敏性等优点,被广泛应用于食物过敏原的检测。本文主要从定性和定量2方面介绍了质谱技术在食品过敏原检测中的研究进展。相关研究对牛乳、鸡蛋、小麦和榛子(坚果类)等主要食物过敏原均有涉及。在定性研究中,以肽质量指纹图谱法和肽碎片离子鉴定法为主,鉴定食物中的过敏原蛋白。在定量研究中,通过标记/无标记技术,也能够实现对微量的目标蛋白进行相对/绝对定量。质谱技术应用于食物过敏原检测中,将有助于提升过敏原检测能力,降低食物过敏安全风险。