Specific detection of potentially allergenic kiwifruit in foods using polymerase chain reaction

Kiwifruit (Actinidia deliciosa and Actinidia chinensis) is allergenic to sensitive patients, and, under Japanese regulations, it is one of the food items that are recommended to be declared on food labeling as much as possible. To develop PCR-based methods for the detection of trace amounts of kiwifruit in foods, two primer pairs targeting the ITS-1 region of the Actinidia spp. were designed using PCR simulation software. On the basis of the known distribution of a major kiwifruit allergen (actinidin) within the Actinidia spp., as well as of reports on clinical and immunological cross-reactivities, one of the primer pairs was designed to detect all Actinidia spp. and the other to detect commercially grown Actinidia spp. (i.e., kiwifruit, Actinidia arguta, and their interspecific hybrids) except for Actinidia polygama. The specificity of the developed methods using the designed primer pairs was verified by performing PCR experiments on 8 Actinidia spp. and 26 other plants including fruits. The methods were considered to be specific enough to yield target-size products only from the target Actinidia spp. and to detect no target-size products from nontarget species. The methods were sensitive enough to detect 5-50 fg of Actinidia spp. DNA spiked in 50 ng of salmon testis DNA used as a carrier (1-10 ppm of kiwifruit DNA) and 1700 ppm (w/w) of fresh kiwifruit puree spiked in a commercial plain yogurt (corresponding to ca. 10 ppm of kiwifruit protein). These methods would be expected to be useful in the detection of hidden kiwifruit and its related species in processed foods.     

Comparison of DNA extraction methods and development of duplex PCR and real-time PCR to detect tomato, carrot, and celery in food

Traceability is of particular importance for those persons who suffer allergy or intolerance to some food component(s) and need a strict avoidance of the allergenic food. In this paper, methodologies are described to fingerprint the presence of allergenic species such as carrot, tomato, and celery by DNA detection. Three DNA extraction methods were applied on vegetables and foods containing or not containing the allergens, and the results were compared and discussed. Fast SYBR Green DNA melting curve temperature analyses and duplex PCR assays with internal control have been developed for detection of these allergenic vegetables and have been tested on commercial foods. Spiking food experiments were also performed, assessing that limits of detection (LOD) of 1 mg/kg for carrot and tomato DNA and 10 mg/kg for celery DNA have been reached.     

Detection of potentially allergenic hazelnut (Corylus avellana) residues in food: a comparative study with DNA PCR-ELISA and protein sandwich-ELISA

Allergen detection is of increasing interest for food labeling purposes. A comparative study with a commercial hazelnut-specific PCR-ELISA and a sandwich-type ELISA detecting hazelnut protein was performed to investigate to what extent immunochemical and DNA-based techniques would correlate in the detection of trace amounts of potentially allergenic hazelnut residues. Both methods were highly sensitive and allowed the detection of even <10 ppm of hazelnut in complex food matrixes. The protein-ELISA was highly specific for hazelnut. However, some foods could lead to false-positive results at the 10 ppm level. The PCR-ELISA did not show any cross-reactions with non-hazelnut foods, thus reducing the probability of having false positives at the trace level. Forty-one commercial food products with and without hazelnut components on their labels were analyzed for the presence of hazelnut. Of the 27 products in which hazelnut components were detected, two samples were not identified by the protein-ELISA, and only one sample, namely one white chocolate having <1 ppm of hazelnut protein, was not detected by PCR-ELISA. The good correlation of the results of PCR-ELISA and protein-ELISA suggested that both PCR-based and immunochemical techniques are suitable for reliable detection of potentially allergenic hazelnut residues in foods at the trace level.     

Quantitative detection of hazelnut (Corylus avellana) in cookies: ELISA versus real-time PCR

Hazelnuts (Corylus avellana) are used widely in the food industry, especially in confectionery, where they are used raw, roasted, or in a processed formulation (e.g., praline paste and hazelnut oil). Hazelnuts contain multiple allergenic proteins, which can induce an allergic reaction associated with symptoms ranging from mild irritation to life-threatening anaphylactic shock. To date, immunochemical (e.g., ELISA or dipstick) and PCR-based analyses are the only methods available that can be applied as routine tests. The aim of this study is to make a comparative evaluation of the effectiveness of ELISA and real-time PCR in detecting and correctly quantifying hazelnut in food model systems. To this end, the performances of two commercial ELISAs were compared to those of two commercial and one in-house-developed real-time PCR assays. The results showed that although ELISA seemed to be more sensitive compared to real-time PCR, both detection techniques suffered from matrix effects and lacked robustness with regard to food processing. As these impacts were highly variable among the different evaluated assays (both ELISA and real-time PCR), no firm conclusion can be made as to which technique is suited best to detect hazelnut in (processed) food products. In this regard, the current lack of appropriate DNA calibrators to quantify an allergenic ingredient by means of real-time PCR is highlighted.     

A sensitive sandwich ELISA for the detection of trace amounts of cashew (Anacardium occidentale L.) nut in foods

Trace amounts of cashew nut protein can provoke severe allergic reactions in sensitive patients. Consequently, commercial food processors and regulatory agencies must be vigilant to prevent cashew nut cross-contamination among foods and ensure proper labeling. Toward this end, we have developed a sandwich enzyme-linked immunosorbent (ELISA) to detect the predominant cashew protein fraction (anacardein or cashew major protein, CMP) that can be extracted in aqueous buffer from food matrixes. Protein G-purified goat antiwhole cashew extract IgG and rabbit anti-CMP IgG were used as capture and secondary antibodies, respectively. Immunoadsorption against several nut and seed proteins significantly minimized the inherent cross-reactivity of these reagents. Food samples spiked with cashew flour and CMP were extracted and tested in a sandwich ELISA where standard curves were based on reactivity with CMP. The assay was optimized to detect as little as 20 ng/mL (0.02 ppm) of CMP and was successfully used to quantify CMP, and thus cashew, in various food matrixes.     

Development of a real-time PCR and a sandwich ELISA for detection of potentially allergenic trace amounts of peanut (Arachis hypogaea) in processed foods

Hidden allergens in food products are, especially for peanut-allergic consumers, a serious problem because even low amounts (approximately 200 microg) of peanut can elicit allergic reactions. Undeclared peanut traces can be found in processed food products, because contaminations with peanut during production processes are frequent. To minimize the risk of such cross-contaminations, it is necessary to develop sensitive analytical methods for the detection of hidden allergens in foods. For this approach we developed two peanut-specific assays based on the detection of peanut protein by specific antibodies (sandwich ELISA) and by the detection of peanut-specific DNA (part of the coding region of Ara h 2) by a real-time PCR. Both tests did not show any cross-reactivity with 22 common food ingredients (cereals, nuts, legumes), and the limit of detection is <10 ppm peanut in processed foods. Thirty-three random samples of food products were tested for the presence of peanut to compare both assay types with each other and to evaluate the percentage of foods on the German market that are contaminated with peanut traces. We found that four products (13.3%) without peanut in the list of ingredients contained peanut protein in a range from 1 to 74 ppm peanut protein and that the results of both tests correlated well. The real-time PCR was able to detect one more positive sample than the sandwich ELISA. In conclusion, both assays are sensitive and specific tools for the detection of hidden allergens in processed foods.     

不同处理方法对虾过敏蛋白分子量及抗原性的影响

本文研究不同处理方法对虾过敏蛋白分子量及抗原性的影响。利用酶解、超高压、微波3种方法处理虾过敏蛋白,利用SDS-PAGE对虾过敏蛋白分子量大小进行测定,用OPA法对各种酶解条件下蛋白质的水解度进行测定,间接竞争ELISA法测定各种处理后过敏蛋白抗原性的变化。结果表明蛋白酶处理后虾过敏蛋白特征条带消失,而经超高压和微波处理的分子量大小没有变化;间接竞争ELISA检测表明三种处理均会导致虾过敏蛋白致敏性不同程度降低或消失。     

Indirect competitive ELISA for determination of traces of peanut (Arachis hypogaea L.) protein in complex food matrices

An indirect competitive ELISA was developed allowing the detection of hidden peanut protein residues down to 2 ppm (micorgrams per gram) in various foods. The high-titer, peanut-specific polyclonal antiserum used recognized potentially allergenic proteins in both native and roasted peanuts. In the absence of a food matrix, extractable protein from roasted peanuts was detected at 104 +/- 13%. From various food items, peanut protein at > or =13 ppm was recovered between 84 and 126%, and at 2 ppm of peanut protein recovery was 143 +/- 6%. Intra- and interassay precision was <15%. In 5 of 17 commercial food products without declaration of peanut components, between 2 and 18 ppm of peanut protein was detected. This is the first assay based on commercially available reactants that allows the reliable determination of trace amounts of hidden peanut allergens in a variety of complex food matrices.     

Qualitative and quantitative evaluation of the genomic DNA extracted from GMO and non-GMO foodstuffs with four different extraction methods

The presence of DNA in foodstuffs derived from or containing genetically modified organisms (GMO) is the basic requirement for labeling of GMO foods in Council Directive 2001/18/CE (Off. J. Eur. Communities 2001, L1 06/2). In this work, four different methods for DNA extraction were evaluated and compared. To rank the different methods, the quality and quantity of DNA extracted from standards, containing known percentages of GMO material and from different food products, were considered. The food products analyzed derived from both soybean and maize and were chosen on the basis of the mechanical, technological, and chemical treatment they had been subjected to during processing. Degree of DNA degradation at various stages of food production was evaluated through the amplification of different DNA fragments belonging to the endogenous genes of both maize and soybean. Genomic DNA was extracted from Roundup Ready soybean and maize MON810 standard flours, according to four different methods, and quantified by real-time Polymerase Chain Reaction (PCR), with the aim of determining the influence of the extraction methods on the DNA quantification through real-time PCR.     

Specific detection of potentially allergenic peach and apple in foods using polymerase chain reaction

Two PCR methods were developed for specific detection of the trnS-trnG intergenic spacer region of Prunus persica (peach) and the internal transcribed spacer region of Malus domestica (apple). The peach PCR amplified a target-size product from the DNA of 6 P. persica cultivars including 2 nectarine and 1 flat peach cultivar, but not from those of 36 nontarget species including 6 Prunus and 5 other Rosaceae species. The apple PCR amplified a target-size product from the DNA of 5 M. domestica cultivars, but not from those of 41 nontarget species including 7 Maloideae and 9 other Rosaceae species. Both methods detected the target DNA from strawberry jam and cookies spiked with peach and apple at a level equivalent to about 10 μg of total soluble proteins of peach or apple per gram of incurred food. The specificity and sensitivity were considered to be sufficient for the detection of trace amounts of peach or apple contamination in processed foods.     

Reliable enzyme-linked immunosorbent assay for the determination of soybean proteins in processed foods

Among allergenic foods, soybean is known as a food causing adverse reactions in allergenic patients. To clarify the validity of labeling, the specific and sensitive detection method for the analysis of the soybean protein would be necessary. The p34 protein, originally characterized to be p34 as an oil-body associated protein in soybean, has been identified as one of the major allergenic proteins and named Gly m Bd 30K. A novel sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of the soybean protein in processed foods was developed using polyclonal antibodies raised against p34 as a soybean marker protein and the specific extraction buffer for extract. The developed sandwich ELISA method was highly specific for the soybean protein. The limit of detection (LOD) and the limit of quantification (LOQ) of the developed ELISA were 0.47 ng/mL (equivalent to 0.19 microg/g in foods) and 0.94 ng/mL (equivalent to 0.38 microg/g in foods), respectively. The recovery ranged from 87.7 to 98.7%, whereas the intra- and interassay coefficients of variation were less than 4.2 and 7.5%, respectively. This study showed that the developed ELISA method is a specific, precise, and reliable tool for the quantitative analysis of the soybean protein in processed foods.     

Quantitative sandwich ELISA for determination of traces of hazelnut (Corylus avellana) protein in complex food matrixes.

A hazelnut-specific sandwich-type ELISA based on polyclonal antisera was developed for detection of hidden hazelnut protein residues in complex food matrixes. In the absence of a food matrix, extractable protein from different native and toasted hazelnuts was detected at rates of 94 +/- 13 and 96 +/- 7% applying standards prepared from native and toasted hazelnuts, respectively. From complex food matrixes, 0.001-10% of hazelnut was recovered between 67 and 132%, in average by 106 +/- 17%. Depending on the food matrix, hazelnut protein could be detected down to the ppb (ng/g) level. Intraassay precision was <6% for hazelnut >/= 0.001% and interassay precision was <15% for hazelnut >/= 0.01%. In 12 of 28 commercial food products without labeling or declaration of hazelnut components, between 2 and 421 ppm of hazelnut protein was detected, demonstrating a remarkable presence of potentially allergenic hazelnut protein "hidden" in commercial food products.     

Sandwich immunoassays for the determination of peanut and hazelnut traces in foods

People suffering from food allergies are dependent on accurate food labeling, as an avoidance diet is the only effective countermeasure. Even a small amount of allergenic protein can trigger severe reactions in highly sensitized patients. Therefore, sensitive and reliable tests are needed to detect potential cross-contamination. In this paper two fast sandwich immunoassays are described for the determination of peanut (Arachis hypogaea) and hazelnut (Corylus avellana) traces in complex food matrices. Mouse monoclonal antibodies were used as capture antibodies, and labeled rabbit polyclonal antibodies were used as detection antibodies in both assays. The assay time was 30 min in total, and cross-reactivities against a variety of fruits and seeds were found to be in the low 10(-4)% (ppm) level or in some cases not detectable. The recoveries in all tested food matrices ranged from 86 to 127%, and the limits of detection were in the range of 0.2-1.2 mg/kg (ppm) in food for both peanut and hazelnut, respectively.     

Detection of allergenic ingredients using real-time PCR: a case study on hazelnut (Corylus avellena) and soy (Glycine max)

Compliance with the European allergen labeling legislation (Directive 2007/68/EC) is only possible when coupled with appropriate methods to detect allergens in food. The aim of the current study was to develop new real-time PCR assays for the detection of hazelnut and soy and evaluate these assays via comparison with commercially available kits. Although the new assays were not as sensitive as the commercial qualitative assays, they proved to be more specific. Moreover, the cross-reactivity study indicated contamination of some of the food products used with either hazelnut or soy, which presents a risk for the allergic consumer. The assays were able to quantify as few as 5-15 genome copies. This unit, used to express analytical results for allergen detection by means of PCR, needs to be converted to a unit expressing the amount of allergenic ingredient in order to be informative. This study emphasizes that the use of real-time PCR for allergen quantification is complicated by the lack of appropriate reference materials for allergens.     

New method of DNA isolation from two food additives suitable for authentication in polymerase chain reaction assays

Locust bean gum and guar gum are galactomannans used as additives (E 410 and E 412, respectively) in the food industry as stabilizing agents. Analytical discrimination between the two additives in gums and foods is now feasible by molecular techniques. However, only complex and time-consuming DNA isolation protocols are available to date. We have developed simple improved protocols to obtain enough DNA suitable for PCR amplification from a few milligrams of commercial E 410 and E 412 additives (containing more than 75% polysaccharides). The suspension of additives in water or 10 mM Tris-HCl, pH 8.5, efficiently recovers DNA suitable for authentication in PCR assays. However, the Tris method was much more efficient for the extraction of DNA from E 410 than for E 412 additives. Conversely, the water method was the most suitable for detecting DNA extracted from E 412 or from E 410/E 412 mixtures. Combined with the use of the two specific ribosomal primer pairs previously designed, our methods are well-suited for a fast and simple high-throughput sample treatment of commercial gums for molecular certification.     

Simultaneous detection of eight food allergens using optical thin-film biosensor chips

Food allergies are important food safety issues nowadays. To maintain the safety of people who experience allergic reactions, labeling is required in many countries and efficient and reliable detection methods are necessary. This paper reports a novel method for the rapid identification of food allergens through the use of a silicon-based optical thin-film biosensor chip with which color change results can be perceived by the naked eye without any extra equipment. The whole system can detect eight food allergens including soybean, wheat, peanut, cashew, shrimp, fish, beef, and chicken simultaneously. Sensitive and specific detection of the absolute detection limit of this method was 0.5 pg of cashew DNA, and the practical detection limit of 0.001%. The biosensor chip detection time was about 30 min after PCR amplification. The assay is proposed as a sensitive, specific, high-throughput, and ready-to-use analytical tool to detect the presence or confirm the absence of eight food allergens.     

Almond allergens: molecular characterization, detection, and clinical relevance

Almond ( Prunus dulcis ) has been widely used in all sorts of food products (bakery, pastry, snacks), mostly due to its pleasant flavor and health benefits. However, it is also classified as a potential allergenic seed known to be responsible for triggering several mild to life-threatening immune reactions in sensitized and allergic individuals. Presently, eight groups of allergenic proteins have been identified and characterized in almond, namely, PR-10 (Pru du 1), TLP (Pru du 2), prolamins (Pru du 2S albumin, Pru du 3), profilins (Pru du 4), 60sRP (Pru du 5), and cupin (Pru du 6, Pru du γ-conglutin), although only a few of them have been tested for reactivity with almond-allergic sera. To protect sensitized individuals, labeling regulations have been implemented for foods containing potential allergenic ingredients, impelling the development of adequate analytical methods. This work aims to present an updated and critical overview of the molecular characterization and clinical relevance of almond allergens, as well as review the main methodologies used to detect and quantitate food allergens with special emphasis on almond.     

Molecular cloning of tropomyosins identified as allergens in six species of crustaceans

Although tropomyosin is known to be a major allergen of crustaceans, its structural information is limited to only five species. In this study, tropomyosin was confirmed to be a major allergen in six species of crustaceans (black tiger prawn, kuruma prawn, pink shrimp, king crab, snow crab, and horsehair crab) by immunoblotting. Then, the amino acid sequences of tropomyosins from these crustaceans were elucidated by a cDNA cloning technique. Sequence data for crustacean tropomyosins including the obtained results reveal that fast tropomyosins are contained in shrimps (or prawns) and lobsters, slow tropomyosins in crabs, and both tropomyosins in crayfishes and hermit crabs. Although fast and slow tropomyosins share a high sequence identity (about 90%) with each other, significant differences are observed in specific regions between both tropomyosins.     

Rapid identification of seaweeds in food products by PCR combined with ALF-RFLP and FINS methodologies

In the present study, two methods for the genetic identification of the most important seaweed species used for human consumption were developed. Both are carried out through PCR amplification of an 18S rRNA gene fragment. The first one is based on the phylogenetic analysis of DNA sequences (FINS), while the second is based on length polymorphism and RFLP visualized by means of an ALF system. The main novelty of this work lies in the fact that it allows genetic identification of the main commercial species of seaweed. Moreover, the developed systems can be applied to all kinds of processed products, including those that have undergone intensive transformation, as for instance canned foods. These methodologies also permit the detection of species in complex matrixes where more than one algal species is present. The methods were validated using products manufactured in a pilot plant showing correct functioning. Finally, the methods were applied to 23 commercial samples including some that had been subjected to intensive thermal treatment, allowing the detection of those that were incorrectly labeled (30%). Therefore, these molecular tools can be used for clarifying questions related to the correct labeling and traceability of commercial products that include some seaweeds in their composition.     

Authentication of anglerfish species (Lophius spp) by means of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and forensically informative nucleotide sequencing (FINS) methodologies

Lophius represents the most important genus of the family Lophiidae from a commercial point of view. The main marketing formats of the species included in this genus are tails and cheeks, making impossible the species identification on the basis of their morphological characters. In the present study, two methods based on the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and phylogenetic analysis of DNA sequences [forensically informative nucleotide sequencing (FINS)] were developed to differentiate the seven species contained in the genus Lophius. In both cases, the molecular marker studied was the cytochrome oxidase subunit I gene (COI). The RFLP analysis of the PCR products digested with the endonuclease Mbo I generated species-specific restriction profiles, and the phylogenetic analysis showing a neighbor-joining tree with independent nodes was strongly supported for all of the studied species. These methods were applied to 40 commercial samples, allowing us to detect the samples incorrectly labeled. The fraudulent labeling ratio was higher in processed products (68.75%) than whole fish (31.25%). The species subjected to mislabeling were L. budegassa (68.75%), L. vomerinus (18.75%), and L. piscatorius (12.5%). Therefore, both methodologies can be independently used to authenticate the species belonging to the genus Lophius, being useful to check the fulfillment of labeling regulations of seafood products and to verify the correct traceability of commercial trade and the control of fisheries.