Immunochemical-based method for detection of hazelnut proteins in processed foods

A competitive enzyme-linked immunosorbent assay (ELISA) was developed to detect hazelnut by using polyclonal antibodies generated against a protein extract of roasted hazelnut. No cross-reactivity was observed in tests against 39 commodities, including many common allergens, tree nuts, and legumes. Hazelnut protein standard solutions at 0.45 ng/mL [inhibition concentration (IC80) of the competitive test] were clearly identified by the ELISA. An extraction and quantification method was developed and optimized for chocolate, cookies, breakfast cereals, and ice cream, major food commodities likely to be cross-contaminated with undeclared hazelnut during food processing. No sample cleanup was required when extracts were diluted 10-fold. Recovery results were generated with blank matrixes spiked at 4 levels from 1 to 10 microg/g hazelnut protein. With the developed extraction and sample handling procedure, hazelnut proteins were recovered at 64-83% from chocolate and at 78-97% from other matrixes. A confirmatory technique was developed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western transfer. The developed methods were applied to a small market survey of chocolate products and allowed the identification of undeclared hazelnut in these products.     

Flow analysis methods for the direct ultra-violet spectrophotometric measurement of nitrate and total nitrogen in freshwaters

Allergy to tree nuts represents an acute health problem. Sensitized people can be inadvertently exposed to hidden allergens resulting from cross-contamination of foods. For this reason, reliable and highly sensitive analytical methods are needed to be developed for control and labeling of food ingredients and products. In the present paper we have proposed a new allergen specific sandwich-ELISA for hazelnut operated in optical and electrochemical modes. The ELISA was based on chicken egg yolk antibodies raised against a major hazelnut allergen, Cor a 9. The developed ELISA has a limit of detection in phosphate buffer of 4 ng mL(-1). No significant cross-reactivity with peanut, wheat or other food ingredients has been detected. Extracts of blank control cookies did not show any false positive response and the limit of detection in cookies was estimated to be 0.1 μg of hazelnut protein per g of food (0.1 ppm). The ELISA protocol was successfully adapted to operate in electrochemical mode and it was applied for the detection of hazelnut traces in cookies.     

Commercial lateral flow devices for rapid detection of peanut (<Emphasis Type="Italic">Arachis hypogaea)</Emphasis> and hazelnut (<Emphasis Type="Italic">Corylus avellana)</Emphasis> cross-contamination in the industrial production of cookies

Lateral flow devices (LFDs) are qualitative immunochromatographic tests for the rapid and specific detection of target analytes. We investigated commercially available LFDs for their ability to detect potentially allergenic peanut and hazelnut in raw cookie dough and chocolate, two important food matrices in the industrial production of cookies. Each three commercial LFDs for the detection of hazelnut and peanut were performed according to the manufacturers’ instructions. All LFDs had comparably satisfactory specificity that was investigated with a variety of characteristic foods and food ingredients used in the production of cookies. In concordance with hazelnut-specific enzyme-linked immunosorbent assays (ELISAs), walnut was the most cross-reactive food for hazelnut-specific LFD. The sensitivity was verified in raw cookie doughs and chocolates that were either spiked with peanut or hazelnut between 1 and 25 mg/kg, respectively. Two hazelnut-specific LFDs detected hazelnut at a level of 3.5 mg/kg in both matrices, whereas the third LFD detected hazelnut at a level of 3.9 mg/kg in dough and 12.5 mg/kg in chocolate. Two peanut-specific LFDs detected peanut at a level of 1 mg/kg in both matrices. The third LFD detected peanut at a level of 14.2 mg/kg in chocolate and 4 mg/kg in dough. In conclusion, specific and sensitive LFD were identified for each hazelnut and peanut, having a level of sensitivity that is comparable to commercial ELISA for the investigated matrices. Such sensitive, specific, and rapid tests are useful analytical tools for allergen screening and sanitation in the industrial manufacture of foods.     

Determination of egg proteins in food products by enzyme immunoassay

An enzyme-linked immunosorbent assay (ELISA) was developed to determine the presence of egg proteins in foods. The polyclonal antibodies developed were specific to whole egg proteins and did not cross-react with any of the 38 nuts, legumes, or other common food ingredients tested. The concentrations of egg proteins that will inhibit 50% of antibody-antigen binding, IC50, were 3-7 ng/mL, and the linear range was 0.5-62.5 ng/mL. The detection limit was 0.2 ppm for various foods. Recoveries ranged from 67 to 96%. The intra- and inter-assay coefficients of variation in this procedure were 10-13% for ice cream spiked at 0.8 and 1.6 ppm. The ELISA has been applied to ice creams, noodles, pasta, and breads. Egg proteins were identified in all declared egg products, and no false positives were found.     

Immunochemical analytical methods for the determination of peanut proteins in foods

Peanut proteins can cause allergenic reactions that can result in respiratory and circulatory effects in the body sometimes leading to shock and death. The determination of peanut proteins in foods by analytical methods can reduce the risk of serious reactions in the highly sensitized individual by allowing for the detection of these proteins in a food at various stages of the manufacturing process. The method performance of 4 commercially available enzyme-linked immunosorbent assay (ELISA) kits was evaluated for the detection of peanut proteins in milk chocolate, ice cream, cookies, and breakfast cereals: ELISA-TEK Peanut Protein Assay, now known as "Bio-Kit" for peanut proteins, from ELISA Technologies Inc.; Veratox for Peanut Allergens from Neogen Corp.; RIDASCREEN Peanut Kit from R-Biopharm GmbH; and ProLisa from Canadian Food Technology Ltd. The 4 test kits were evaluated for accuracy (recovery) and precision using known concentrations of peanut or peanut proteins in the 4 food matrixes. Two different techniques, incurred and spiked, were used to prepare samples with 4 known concentrations of peanut protein. Defatted peanut flour was added in the incurred samples, and water-soluble peanut proteins were added in the spiked samples. The incurred levels were 0.0, 10, 20, and 100 microg whole peanut per g food; the spiked levels were 0.0, 5, 10, and 20 microg peanut protein per g food. Performance varied by test kit, protein concentration, and food matrix. The Veratox kit had the best accuracy or lowest percent difference between measured and incurred levels of 15.7% when averaged across all incurred levels and food matrixes. Recoveries associated with the Veratox kit varied from 93 to 115% for all food matrixes except cookies. Recoveries for all kits were about 50% for cookies. The analytical precision, as measured by the variance, increased with an increase in protein concentration. However, the coefficient of variation (CV) was stable across the 4 incurred protein levels and was 7.0% when averaged across the 4 food matrixes and analytical kits. The R-Biopharm test kit had the best precision or a CV of 4.2% when averaged across all incurred levels and food matrixes. Because measured protein values varied by test kit and food matrix, a method was developed to normalize or transform measured protein concentrations to an adjusted protein value that was equal to the known protein concentration. The normalization method adjusts measured protein values to equal the true protein value regardless of the type test kit or type food matrix.     

Determination of egg proteins in snack food and noodles

Egg is one of the 5 major allergenic foods that are responsible for more than 3/4 of food allergies in children. Food-allergic responses can be controlled by avoidance of the offending foods. The applicability of a commercial enzyme-linked immunosorbent assay (ELISA) kit for the detection of egg in food products such as cookies, crackers, pretzels, salad dressings, and raw and cooked noodles was evaluated. A preliminary evaluation of an antibody-based biosensor was also performed. A National Institute of Standards and Technology (NIST) whole dried egg powder reference material, SRM 8415, was used as a standard. A homogeneous and stable aqueous egg suspension was prepared for the evaluation of the performance of the Veratox for Egg Allergen Test (Neogen Corp., Lansing, MI). This test does not detect egg yolk proteins. Each gram of the aqueous dried egg suspension contained 643 microg whole dried egg, 0.5 mg thimerosal, and 2.5 mg bovine serum albumin. When cookies, crackers, salad dressings, noodles, and ice cream were spiked at a level of 24 mg/kg SRM 8415, recoveries for whole egg averaged about 28%. All foods containing egg as indicated on the ingredient label were found positive by the Veratox test. No false positives occurred in samples that did not contain eggs. Similar results were obtained using the Naval Research Laboratory (NRL) array biosensor, an evanescent wave fluoroimmunosensor. Results for cooked noodles showed that they contained <1% of the egg found in uncooked noodles. A comparison of extracts from cooked and uncooked noodles by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed differences in protein profiles. The boiling of the noodles could have reduced the immunoreactivity of the egg proteins to the antibodies used in the kit or rendered the egg proteins nonextractable.     

Extractability, stability, and allergenicity of egg white proteins in differently heat-processed foods

Hen's egg white protein is a major cause of food allergy, and a considerable number of countries have introduced labeling directions for processed food products. To control compliance with these regulations, analytical assays for the detection of egg in manufactured foods have been developed. In this study, we have tested the performance of 3 commercially available kits for quantitative egg analysis using 6 model heat-processed foods. The 3 assays worked well under standard conditions with soluble egg white proteins, but only the kit using a denaturing-reducing extraction buffer detected egg in complex heat-treated food matrixes. The differently extracted food samples were further used to evaluate the stability and allergenicity of the egg white allergens ovalbumin, ovomucoid, ovotransferrin, and lysozyme with polyclonal anti-egg antibodies and sera of 6 patients with egg allergy. It could be shown that differences in egg protein extractability have a significant impact on the interpretation of study results.     

Marker peptide selection for the determination of hazelnut by LC–MS/MS and occurrence in other nuts

The aim of this work was identifying and selecting hazelnut marker peptides and subsequently developing a complementary method of common immunoassay for the detection of hazelnut. For this purpose, at first, an in silico digestion of three major hazelnut allergens (Cor a 8, Cor a 9 and Cor a 11) was performed to get information about expected peptides. After extraction and trypsin digestion of hazelnut proteins, the samples were measured with tandem mass spectrometry (MS/MS) by direct infusion, which led to identification of 14 peptides. Eight of them with the highest MS signal were synthesized and used as standards for developing a liquid chromatography (LC)–MS/MS method in selected reaction monitoring (SRM) mode. Since almost all food allergens derived from nuts belong to the seed storage protein family and have homologue structure, a Basic Local Alignment Search Tool (BLAST) search was performed to identify the hazelnut specificity of the developed method. According to BLAST, only one peptide occurs in three other nuts, and the remaining seven selected peptides are hazelnut specific. Additionally to hazelnut, the eight other listed nuts in Directive 2003/89/EC as allergen were extracted, digested and measured with the developed method to prove the BLAST results. The analytical data confirmed that six peptides are hazelnut specific, on the contrary to anti-hazelnut antibodies, which showed cross-reactivities to all other nut extracts. Comparing these results, it could be shown that with this LC–MS/MS method in SRM mode, the specific detection of hazelnut is possible.     

Sensitive and specific detection of potentially allergenic almond (Prunus dulcis) in complex food matrices by Taqman(®) real-time polymerase chain reaction in comparison to commercially available protein-based enzyme-linked immunosorbent assay

Currently, causative immunotherapies are lacking in food allergy. The only option to prevent allergic reactions in susceptible individuals is to strictly avoid the offending food. Thus, reliable labelling of allergenic constituents is of major importance, but can only be achieved if appropriate specific and sensitive detection techniques for foods with allergenic potential are available. Almond is an allergenic food that requires mandatory labelling on prepackaged foods and belongs to the genus Prunus. Species of this genus are phylogenetically closely related. We observed commercially available almond specific ELISA being highly cross-reactive with other foods of the Prunoideae family, resulting in a false-positive detection of up to 500,000 mg kg(-1) almond. Previously published PCR methods were reported to be cross-reactive with false positive results >1200 mg kg(-1). We describe the development of a novel almond specific real-time PCR, based on mutated mismatch primers and sequence specific Taqman(?) probe detection, in comparison with two quantitative commercially available ELISA. PCR sensitivity was investigated with chocolate, chocolate coating and cookies spiked between 5 and 100,000 mg kg(-1) almond. In all matrices almond was reproducibly detected by real-time PCR at the lowest spike level of 5 mg kg(-1). Further, between 100 and 100,000 mg kg(-1) spiked almond, the method featured good correlation between quantified copy numbers and the amount of spiked almond. Within this range a similar relation between detectable signal and amount of almond was observed for both PCR and ELISA. In contrast to ELISA the Taqman(?) real-time PCR method was highly specific in 59 food items with negligible cross-reactivity for a very limited number of Prunoideae foods. The real-time PCR analysis of 24 retail samples was in concordance with ELISA results: 21% (n=5) contained undeclared almond. This is the first completely disclosed real-time PCR method for a specific and potentially quantitative almond detection. This PCR method detects almond at a level where severe allergic reactions should not be expected for the majority of the almond allergic individuals.     

ELISA kit for mustard protein determination: interlaboratory study

An interlaboratory study in 12 laboratories was performed to prove the validation of the ELISA method developed for the quantitative determination of mustard protein in foods. The ELISA kit used for this study is based on rabbit polyclonal antibody. This kit did not produce any false-positive results or cross-reactivity with in-house validation for a broad range of food matrixes with no detectable mustard protein. All participants obtained the Mustard ELISA kit with standard operational procedures, a list of samples, samples, and a protocol for recording test results. The study included 15 food samples and two spiked samples. Seven food matrix samples of zero mustard content and four samples with mustard declared as an ingredient showed mustard protein content lower than that of the first standard (0.42 mg/kg). Four samples with mustard declared as an ingredient revealed mustard protein content above 12.5 mg/kg (the highest standard). The statistical tests (Cochran, Dixon, and Mandel) and analysis of variance were used to evaluate the interlaboratory study results. Repeatability and reproducibility limits, as well as an LOQ (1.8 mg mustard proteins/kg) and LOD (0.5 mg mustard proteins/kg), for the kit were calculated.     

Assessing hazelnut allergens by protein- and DNA-based approaches: LC-MS/MS,ELISA and real-time PCR

Hazelnut (Corylus avellana L.) is responsible for a significant part of the allergies related to nuts. Still, it is a very much appreciated nut and as consequence is widely used in all types of processed foods, such as chocolates. Correct food labelling is currently the most effective means of preventing the consumption of allergenic ingredients, namely hazelnut, by the sensitised/allergic individuals. Thus, to verify labelling compliance and to ensure allergic patient protection, the development of highly sensitive methodologies is of extreme importance. In this study, three major methodologies, namely enzyme-linked immunosorbent assays (ELISA), liquid chromatography coupled with mass spectrometry and real-time polymerase chain reaction, were evaluated for their performance regarding the detection of hazelnut allergens in model chocolates. The sandwich ELISA and respective antibodies were in-house developed and produced. With sensitivity levels of approximately 1 mg kg^?1 and limits of quantification of 50–100 mg kg^?1, all the performed methods were considered appropriate for the identification of hazelnut in complex foods such as chocolates. To our knowledge, this was the first successful attempt to develop and compare three independent approaches for the detection of allergens in foods.

SYBR green real-time PCR used to detect celery in food

Celery was found to provoke human allergenic response in some countries. Labeling of celery ingredients was required by the European Union, and the threshold set at 10 mg/kg (0.001%). In our study, a celery mannitol transporter (Mat3) gene-based detection method was established by means of SYBR Green real-time PCR technique. No cross-reactivity was found between celery and the other food materials. Absolute detection limit (LODa), relative detection limit (LODr), and practical detection limit (LODp) of the method were determined through experiments on pure celery DNA, DNA mix, and spiked food samples. The method was able to detect 0.001% raw food sample and 0.01% heated food sample. The utility of the method was confirmed by the investigation of 13 commercial foods.     

Food allergen detection with biosensor immunoassays

An optical biosensor was used to develop both direct and sandwich immunoassays for the detection of proteins from milk, egg, hazelnut, peanut, shellfish, and sesame in food samples. Affinity-purified polyclonal antibodies raised against the proteins were immobilized on the biosensor chip. Food samples were injected and the proteins that bound to the antibodies on the surface were detected by a shift in the resonance angle. By adding a second antibody in a sandwich assay, matrix effects could be overcome and the sensitivity and selectivity enhanced. Detection of allergen levels down to 1-12.5 microg/g in food samples was demonstrated for the various assays. Good agreement of results was also obtained from parallel analysis with alternative immunoassays, including rocket immunoelectrophoresis, enzyme immunoassay, and immunoblotting. The present study demonstrates that the sensitivity of the described biosensor technique is comparable to the most sensitive enzymed-linked immunosorbent assays.     

Sensitive and specific detection of potentially allergenic almond (Prunus dulcis) in complex food matrices by Taqman real-time polymerase chain reaction in comparison to commercially available protein-based enzyme-linked immunosorbent assay

Currently, causative immunotherapies are lacking in food allergy. The only option to prevent allergic reactions in susceptible individuals is to strictly avoid the offending food. Thus, reliable labelling of allergenic constituents is of major importance, but can only be achieved if appropriate specific and sensitive detection techniques for foods with allergenic potential are available. Almond is an allergenic food that requires mandatory labelling on prepackaged foods and belongs to the genus Prunus. Species of this genus are phylogenetically closely related. We observed commercially available almond specific ELISA being highly cross-reactive with other foods of the Prunoideae family, resulting in a false-positive detection of up to 500,000 mg kg⁻¹ almond. Previously published PCR methods were reported to be cross-reactive with false positive results >1200 mg kg⁻¹.We describe the development of a novel almond specific real-time PCR, based on mutated mismatch primers and sequence specific Taqman^® probe detection, in comparison with two quantitative commercially available ELISA. PCR sensitivity was investigated with chocolate, chocolate coating and cookies spiked between 5 and 100,000 mg kg⁻¹ almond. In all matrices almond was reproducibly detected by real-time PCR at the lowest spike level of 5 mg kg⁻¹. Further, between 100 and 100,000 mg kg⁻¹ spiked almond, the method featured good correlation between quantified copy numbers and the amount of spiked almond. Within this range a similar relation between detectable signal and amount of almond was observed for both PCR and ELISA. In contrast to ELISA the Taqman^® real-time PCR method was highly specific in 59 food items with negligible cross-reactivity for a very limited number of Prunoideae foods. The real-time PCR analysis of 24 retail samples was in concordance with ELISA results: 21% (n = 5) contained undeclared almond. This is the first completely disclosed real-time PCR method for a specific and potentially quantitative almond detection. This PCR method detects almond at a level where severe allergic reactions should not be expected for the majority of the almond allergic individuals.     

Reliable detection of milk allergens in food using a high-resolution, stand-alone mass spectrometer

Reliable methods are needed for detection of allergenic milk proteins in complex food matrixes. The feasibility of an LC/high-resolution MS method for the analysis of milk proteins in a thermally processed model food (incurred cookies) and in white wine spiked, respectively, with milk powder and caseinate is described. Detection of milk proteins was based on the identification of unique peptides in the tryptic digests of cookie/wine extracts using an RP-HPLC separation coupled to an Exactive nonhybrid mass spectrometer using Orbitrap technology. The extremely high mass accuracy and resolution provided by the Orbitrap analyzer allowed a fast preliminary identification of four previously proposed peptide markers of caseins using only accurate values of the m/z of their ions. No interference was observed, despite the complexity of the analyzed matrixes. Moreover, the availability of a high- energy, collisionally activated dissociation cell integrated in the mass spectrometer enabled acquisition of peptide MS/MS-like spectra through post-source fragmentation. Confirmation of peptide marker identity could then be achieved by a comparison between experimental and predicted product ions. The described method shows the great potential of Orbitrap MS as a reliable technique in the field of protein allergen detection once the peptide markers are identified.     

Detection of hidden hazelnut protein in food by IgY-based indirect competitive enzyme-immunoassay

The development of an indirect competitive enzyme-immunoassay for the detection of hidden hazelnut protein in complex food matrices is described. A sensitive and selective polyclonal antibody was raised by immunisation of laying hens with protein extracts from roasted hazelnuts. In contrast to traditional antibody generation in mammals, the antibody was not isolated from the blood of immunised mammals but from the egg yolk of immunised chickens. A standard calibration curve was optimised using immunoaffinity purified antibody extract and a coating antigen concentration of 10 μg ml⁻¹. One percent skim milk powder was chosen for blocking. The assay has a minimum detection limit of 10 μg l⁻¹, with an IC₅₀ of 618 μg l⁻¹ when a 50 mM phosphate buffer at pH 7.5 and 10 mM sodium chloride is used as assay buffer. The cross reactivity testing shows a high specificity for hazelnut proteins and various foods and food additives were found to be non reactive except beans, sunflower seed or poppy seed.     

Detection of soy proteins in processed foods: literature overview and new experimental work

Several tests for the detection of soy proteins in foods have been described in the literature, and some are commercially available. This article gives an overview of these methods and discusses the advantages and disadvantages of each individual method. Based on the conclusions of this inventory, an experimental approach was designed to improve the sensitivity of measuring soy protein in processed foods. The aimed sensitivity is 10 ppm (10 microg soy protein in 1 g solid sample), which is over 100-fold lower than presently available tests. The aimed sensitivity is this low because levels of food allergens at 10 ppm and above may provoke reactions in food allergic persons. Native soybean meal, soy protein isolate, soy protein concentrate, and textured soy flakes were used as test materials. Several extraction procedures were compared and a new method using high pH was selected. Polyclonal antibodies were raised in rabbits and goats, and immunopurified antibodies were used in sandwich and inhibition enzyme-linked immunosorbent assay (ELISA). Extraction at pH 12 resulted in good yields for all tested samples, both quantitatively (Bradford) and qualitatively by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunopurified rabbit antibodies against this extract used in a competition ELISA format resulted in a sensitive test with a detection limit of 0.02 microg/mL, corresponding to 0.4 microg/g (0.4 ppm) in food samples. Cross-reactivity with some main food ingredients was measured and appeared to be negative in all cases. The presently developed test is applicable for soy ingredients and soy-containing foods that are processed in different ways. The limit of quantitation is 1 ppm, which is an enormous improvement over earlier described methods.     

Development and validation of a sandwich ELISA for the determination of potentially allergenic sesame (Sesamum indicum) in food

This paper presents a sandwich enzyme-linked immunosorbent assay (ELISA) that allows the determination of traces of sesame in food. Chicken anti-sesame antibodies, used as coating antibodies, and rabbit anti-sesame antibodies, used as secondary antibodies, were prepared by immunization with a protein extract of white, peeled sesame. The ELISA did not show any cross-reactivity with 19 food ingredients commonly found in sesame-containing foodstuffs such as seeds, nuts, and cereals. In whole grain bread, crisp toast, and snacks, the limit of detection (S/N = 3) was 0.5, 0.5, and 0.3 μg sesame protein/g, and the limit of quantification (S/N = 10) was 0.6, 0.8, and 1.4 μg sesame protein/g, respectively. The analysis of blank food matrices (whole grain bread, white bread, crisp toast, and snacks) spiked with sesame protein at four spike levels generally resulted in mean recoveries from 72% to 145%. In the case of spiking blank food matrices with sesame seeds, the ELISA proved to be more accurate for whole wheat cookies than for whole wheat bread.     

Production and characterization of antibodies against crosslinked gelatin nanoparticles and first steps toward developing an ELISA screening kit

Nanotechnologies are finding a growing range of applications in the food sector. Nanoparticles are used notably to add vitamins and other nutrients to foods and beverages without affecting taste and color. They are also used to develop new tastes, preserve food texture, control the release of flavors, improve the bioavailability of compounds such as antioxidants and vitamins, and monitor freshness with nanosensors. Crosslinked gelatin nanoparticles are a component of nano-sized carriers for nutrient and supplement delivery in foods and related products. This paper describes the production and characterization of polyclonal antibodies against gelatin nanoparticles. Two immunization schemes were investigated: subcutaneous injection with and without a first intravenous injection. Two enzyme-linked immunosorbent assay formats were used to characterize the antibodies: an inhibition format with an antigen-coated plate for detection of the immune response and a sandwich format for development of the method. The antibodies showed good sensitivity with an IC50 equal to 0.11 ng mL(-1) using indirect ELISA format and a good specificity for the nanomaterials, without significant cross-reactivity against native gelatin. The limit of detection was determined-0.42, 0.27, 0.26, and 0.24 μg mL(-1) for apple, orange juice, milk, and soft drink matrices, respectively. ELISA technology offers rapid, low-cost assays for screening foods, feeds, and beverages. We have studied a prototype ELISA for detection of gelatin-based nanocarrier systems. Fruit juices, milk, and a soft drink were the matrices selected for assay development.     

Detection of cashew nut DNA in spiked baked goods using a real-time polymerase chain reaction method

The detection of potentially allergenic foods, such as tree nuts, in food products is a major concern for the food processing industry. A real-time polymerase chain reaction (PCR) method was designed to determine the presence of cashew DNA in food products. The PCR amplifies a 67 bp fragment of the cashew 2S albumin gene, which is detected with a cashew-specific, dual-labeled TaqMan probe. This reaction will not amplify DNA derived from other tree nut species, such as almond, Brazil nut, hazelnut, and walnut, as well as 4 varieties of peanut. This assay was sensitive enough to detect 5 pg purified cashew DNA as well as cashew DNA in a spiked chocolate cookie sample containing 0.01% (100 mg/kg) cashew.