Walnut Allergens Research Articles

Comprehensive characterization and detection of nut allergens in bakery foods using Q-TOF mass spectrometry and bioinformatics

Abstract Food allergy is a growing health issue worldwide and the demand for sensitive, robust and high-throughput analytical methods is rising. In recent years, mass spectrometry-based methods have been established for multiple food allergen detection. In the present study, a novel method was developed for the simultaneous detection of almond, cashew, peanut, and walnut allergens in bakery foods using liquid chromatography–mass spectrometry. Proteins unique to these four ingredients were extracted, followed by trypsin digestion, quadrupole time-of-flight (Q-TOF) mass spectrometry and bioinformatics analysis. The raw data were processed by de-novo sequencing module plus PEAKS DB (database search) module of the PEAKS software to screen peptides specific to each nut species. The thermal stability and uniqueness of these candidate peptides were further verified using triple quadrupole mass spectrometry (QQQ-MS) in multiple reaction monitoring (MRM) mode. Each nut species was represented by four peptides, all of which were validated for label-free quantification (LFQ). Calibration curves were constructed with good linearity and correlation coefficient (r2) greater than 0.99. The limits of detection (LODs) were determined to range from 0.11 to 0.31 mg/kg, and were compared with the reference doses proposed by Voluntary Incidental Trace Allergen Labelling (VITAL). The recoveries of the developed method in incurred bakery food matrices ranged from 72.5% to 92.1% with relative standard deviations (RSD) of <5.2%. The detection of undeclared allergens in commercial bakery food samples confirmed the presence of these allergens. In conclusion, this method provides insight into the qualitative and quantitative detection of trace levels of nut allergens in bakery foods.

Simultaneous quantification of specific food allergen proteins using a fluorescent multiplex array

The aim was to develop a fluorescent multiplex array for simultaneously measuring regulated food allergens using specific allergen protein molecules from peanut, tree nut, cow’s milk, egg, soy, fish, shellfish, sesame, mustard and celery. Microspheres coupled to specific monoclonal antibodies were used for allergen detection, with purified allergens as reference standards.Standard curves for 17 allergens covered a 5-log dynamic range. Intra- and inter-assay acceptance criteria were within 70–130% recovery and a CV of ≤15%. Food reference materials contained high levels of their respective major allergens (2000–175,000 µg/g), Similar high allergen levels were found in 10 selected foods analysed using a 9-plex array. Egg, milk, peanut, hazelnut and walnut allergens were detectable in chocolate bars with incurred allergens at 3, 10, 30, and 100 ppm. The multiplex array is an efficient tool for measuring specific food allergens, with applications for risk assessment and standardization of therapeutic products for food allergy.

Mass Spectrometry-Based Proteomic Approach in <i>Juglans regia</i>

We proposed a MALDI mass spectrometry based approach to characterize walnut allergens. The strategy is based on the extraction of hydro-soluble tissue proteins followed by protein fractionation and mass-spectrometry analysis. Linear MALDI was adopted to evaluate the intact protein mass information and the presence of glycoproteins.

Survey of Commercial Food Products for Detection of Walnut (Juglans regia) by Two ELISA Methods and Real Time PCR.

Labeling of food allergens in accordance with legal regulations is important to protect the health of allergic consumers. The requirements for detecting allergens in foods involve adequate specificity and sensitivity to identify very small amounts of the target allergens in complex food matrices and processed foods. In this work, one hundred commercial samples were analyzed for walnut detection using three different methods: a sandwich enzyme-linked immunosorbent assay (ELISA) kit based on polyclonal antibodies, a direct ELISA using a recombinant multimeric scFv, and a real time PCR. The most sensitive method was real time PCR followed by sandwich ELISA kit and multimeric scFv ELISA. There was agreement between the three methods for walnut detection in commercial products, except for some heat-treated samples or those that contained pecan. The walnut ELISA kit was less affected by sample processing than was the multimeric scFv ELISA, but there was cross-reactivity with pecan, producing some false positives that must be confirmed by real time PCR. According to the results obtained, 7.0 to 12.6% of samples (depending on the analytical method) contained walnut but did not declare it, confirming there is a risk for allergic consumers. Moreover, there was one sample (3.7%) labelled as containing walnut but that tested negative for this tree nut. Genetic and immunoenzymatic techniques offer complementary approaches to develop a reliable verification for walnut allergen labeling.

Component resolved diagnosis of walnut allergy in young children: Jug r 1 as a major walnut allergen.

Walnuts (WN) are one of the main causes of tree nut allergies. However, the potential value of component resolved diagnosis (CRD) for WN allergy is controversial. We analyzed the clinical and immunological features of clinical WN allergy and the usefulness of CRD in young children. Forty-one participants with a history of ingesting WN who were assessed for serum-specific IgE to WN (WNsIgE) using CRD (ImmunoCAP ISAC 112) at the Department of Pediatrics in Ajou University Hospital were enrolled and their demographic profiles, clinical symptoms, and laboratory findings were evaluated. A total of 32 patients were diagnosed with clinical WN allergy, of which 31 had specific immunoglobulin E antibodies to Jug r 1 (Jug r1-sIgE). The Jug r 1-sIgE levels were higher in WN-allergic patients than in WN-tolerant patients and significantly higher in patients with anaphylaxis than in the WN-tolerant patients. Receiver-operating characteristic curves demonstrated that the Jug r 1-sIgE level was much better in discriminating between clinical WN allergy and WN tolerance in young children than the WN-sIgE level. Jug r 1 is the major component allergen in young children with clinical WN allergy. To measure Jug r 1-sIgE appears to be a promising approach for both diagnosis and predicting severity in young children with a history of suspected WN allergy.

Food allergens could promote the Th2 mediated autoantibody development in an autoimmune skin disease bullous pemphigoid

Abstract Bullous pemphigoid (BP) is the most common autoimmune skin blistering disease and is characterized by the presence of autoantibodies (autoAbs) against skin hemidesmosomal adhesion molecules. The predominant antigenic target is the non-collagenous domain 16 (NC16A) of BP180. AutoAbs against NC16A lead to loss of adhesion and subsequent subepidermal blistering. It is unclear what triggers the development of autoAbs. Food allergens are known to induce T helper 2 (Th2) antibody responses and our previous studies have shown the possible role of environmental antigens/allergens in promoting autoAb development. The goal of this study is to investigate whether Th2 mediated immune responses against food allergens drive the autoAb development in BP. BP serum antibodies were tested against a panel of common environmental and food allergens. Anti-NC16A IgE and IgG4 monoclonal antibodies (mAbs) and their naïve counterparts were generated from a BP patient to determine their specificity or cross-reactivity. We found that antibodies from BP patients recognized food allergens, such as walnut, but not other types of allergens, such as insects, epithelia, fungi, or pollens. The levels of serum IgE and IgG4, but not IgG1, against selected food allergens were significantly higher than controls. The anti-NC16A IgE and IgG4 mAbs cross-reacted to food allergens, and their naïve mAb counterparts had reduced reactivity to NC16A, but still reacted strongly to food allergens. In addition, mice immunized with walnut allergen produced anti-NC16A antibodies. Our results indicate that Th2-associated autoAbs from BP subjects might be linked to antibody responses toward food allergens and may provide a target for the management and treatment of BP.

Sequence analysis of digestion-resistant peptides may be an efficient strategy for studying the linear epitopes of Jug r 1, the major walnut allergen

Jug r 1, the major allergen of walnut, triggers severe allergic reactions through epitopes. Hence, research on the efficient strategy for analyzing the linear epitopes of Jug r 1 are necessary. In this work, bioinformatics analysis was used to predict the linear epitopes of Jug r 1. Overlapping peptide synthesis was used to map linear epitopes. In vitro simulated gastrointestinal digestion and HPLC-MS/MS were used to identify digestion-resistant peptides. The results showed that six predicted linear epitopes were AA28-35, AA42-49, AA55-62, AA65-73, AA97-104, and AA109-121. AA16-30 and AA125-139 were identified by the sera of walnut allergic patients. Five digestion-resistant peptides were AA19-33, AA40-45, AA54-74, AA96-106, and AA117-137. The predicted results only included one of the linear epitopes identified by sera, while the digestion-resistant peptides covered all. Therefore, the digestion-resistant property of food allergens may be a promising direction for studying the linear epitopes of Jug r 1.

WALNUT AND PECAN CROSS-DESENSITIZATION: EPITOPE DETERMINATION

Introduction Clinical trials using oral immunotherapy (OIT) for the treatment of walnut food allergy have shown promising results. In a randomized, double-blind, placebo-controlled, clinical trial participants with walnut and pecan food allergies underwent treatment with walnut OIT (n=12). Participants allergic to both walnut and pecan received only walnut oral immunotherapy. Participants receiving walnut oral immunotherapy passed food challenges against both walnut and pecan, suggesting possible cross-desensitization in individuals with pecan and walnut allergies. We sought to test the hypothesis that cross-desensitization occurs due to a common epitope in walnut and pecan allergens proteins following walnut oral immunotherapy, subjects allergic to walnut and pecan will become desensitized to both allergens. Methods Walnut and pecan proteins were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-Page), blotted onto a nitrocellulose membrane, and probed with plasma and secondary antibodies. IgE and IgG4binding were examined for walnut and pecan allergic plasma at two time points: baseline and 1-2 years post-oral immunotherapy. Results In blots probed with baseline serum and anti-IgE, binding appeared with a 9 kDa walnut allergen protein. As expected this binding was not present in blots probed with post-OIT serum and anti-IgE. This band was not seen in blots probed with baseline serum and anti-IgG4. In blots probed with post OIT and anti-IgG4, binding appeared with 9 Da walnut and pecan allergen proteins. Conclusions Results indicate a common epitope between walnut and pecan allergen proteins. In addition, an IgG4-recognized allergen protein not observed at baseline, unexpectedly appeared following walnut OIT.

Retrospective definition of reaction risk in Italian children with peanut, hazelnut and walnut allergy through component-resolved diagnosis

BackgroundSerum IgE evaluation of peanut, hazelnut and walnut allergens through the use of component-resolved diagnosis (CRD) can be more accurate than IgE against whole food to associate with severe or mild reactions. ObjectivesThe aim of the study was to retrospectively define the level of reaction risk in children with peanut, hazelnut and walnut sensitization through the use of CRD. Methods34 patients [n=22 males, 65%; median age eight years, interquartile range (IQR) 5.0–11.0 years] with a reported history of reactions to peanut and/or hazelnut and/or walnut had their serum analyzed for specific IgE (s-IgE) by ImmunoCAP® and ISAC® microarray technique. ResultsIn children with previous reactions to peanut, the positivity of Arah1 and Arah2 s-IgE was associated with a history of anaphylaxis to such food, while the positivity of Arah8 s-IgE were associated with mild reactions. Regarding hazelnut, the presence of positive Cora9 and, particularly, Cora14 s-IgE was associated with a history of anaphylaxis, while positive Cora1.0401 s-IgE were associated with mild reactions. Concerning walnut, the presence of positive Jug r 1, Jug r 2, Jug r 3 s-IgE was associated with a history of anaphylaxis to such food. ImmmunoCAP® proved to be more useful in retrospectively defining the risk of hazelnut anaphylaxis, because of the possibility of measuring Cor a14 s-IgE. ConclusionsOur data show that the use of CRD in patients with allergy to peanut, hazelnut and walnut could allow for greater accuracy in retrospectively defining the risk of anaphylactic reaction to such foods.

A subset of walnut allergic adults is sensitized to walnut 11S globulin Jug r 4.

The role of sensitization to commercially available allergens of English walnut (Juglans regia) Jug r 1, 2 and 3 in walnut allergy has been previously investigated in walnut allergic adults and was unable to explain all cases of walnut allergy. Identify recognized walnut allergens, other than the ones previously investigated (Jug r 1-3), in walnut allergic adults and determine the sensitization frequency and diagnostic value. Three different in-house walnut extracts were prepared and analysed on SDS-PAGE blots to identify allergenic walnut proteins. Immunoblots and immunoprecipitation, followed by LC-MS analysis, were performed to screen for, and confirm, IgE binding to walnut allergens in selected walnut allergic adults. In a cohort of 55 walnut challenged adults, including 33 allergic and 22 tolerant, sensitization to native and recombinant walnut allergen Jug r 4 was assessed using immunoblotting and immuno-line blot (EUROLINE), respectively. Screening of sera of 8 walnut allergic adults identified Jug r 4 as an allergen in our population. In the total cohort of 55 subjects, 5 were positive for Jug r 4 on immunoblot and 10 on EUROLINE. All but one EUROLINE positive subject had a positive food challenge (sensitivity 27%, specificity 95%, PPV 90%, NPV 47%). All 5 subjects positive on immunoblot were also positive on EUROLINE. LC-MS analysis showed a lack of Jug r 4 in the ImmunoCAP extract. Co-sensitization to other 11S albumins (eg hazelnut Cor a 9) was common in Jug r 4 sensitized subjects, potentially due to cross-reactivity. Walnut 11S globulin Jug r 4 is a relevant minor allergen, recognized by 27% of walnut allergic adults. It has a high positive predictive value of 90% for walnut allergy. Specific IgE against Jug r 4 occurred mostly with concomitant sensitization to other walnut components, mainly Jug r 1.

Identification and implication of an allergenic PR-10 protein from walnut in birch pollen associated walnut allergy.

English walnut (Juglans regia) belongs to the most important allergenic tree nuts. Co-sensitization with birch (Betula verrucosa) pollen has been reported. We aimed to identify a walnut allergen homologous to the major birch pollen allergen Betv1. A cDNA encoding a Betv1-homologous allergen (Jugr5) in walnut kernels was cloned by RT-PCR. Jugr5 was expressed in Escherichia coli, purified by column chromatography and characterized by circular dichroism spectroscopy. Specific IgE levels to walnut, Betv1, and Jugr5 in birch pollen allergics (n = 16) with concomitant walnut allergy were measured by ImmunoCAP: 44% of the patients were tested positive to walnut while 94% were reactive to Jugr5, and 100% to Betv1. Jugr5 and Betv1 allergens showed bidirectional IgE cross-reactivity by competitive ELISA and were capable of inducing histamine release from effector cells. Immunoblot competition experiments demonstrated the presence of IgE-reactive Jugr5 in walnut extract, but at low levels. A Betv1-like allergen was identified in walnut. Diagnostic use of Jugr5 will compensate for the low sensitivity of walnut extract for patients with birch pollen associated walnut allergy.

The allergenic potential of walnuts treated with high pressure and heat in a mouse model of allergy

Abstract This study investigates the effects of high hydrostatic pressure (HHP) and/or thermal treatment on walnut immunoreactivity. The residual immunoreactivity of processed walnuts was assessed by competitive inhibition enzyme-linked immunosorbent assays (ci-ELISA). Compared to HHP or boiling treatment, walnut allergens (WA) subjected to HHP and thermal treatment at 650 MPa, 100 °C for 15 min (WAH), were able to diminish the IgE binding capacity more efficiently with the highest reduction up to 86.37%. The effects of processed walnut on the BALB/c mouse model of allergy were characterized using IgE binding capacity, histamine levels in serum, cytokine mRNA expressions. The results showed that significant reductions of 41.04% and 19.76% were observed in serum specific IgE binding capacity and histamine levels, respectively, in mice fed with WAH, accompanied by a repression of both IL-4 (36.79%) and IFN-γ (34.39%) mRNA expressions in ileums. Consequently, clinical signs for allergic reactions were significantly alleviated in WAH treated mice, which indicated that the combination of HHP and thermal treatment HHP could be a potential strategy for the reduction of walnut allergens. Industrial relevance Allergic reactions to walnuts were serious. High hydrostatic pressure (HHP) processing was reported to decrease allergenic activity in rice, fish, milk, peanut and celery through changing allergen conformation. In the study, high pressure and thermal were used to reduce the allergic properties of walnut. Furthermore, a BALB/c mouse model of walnut allergy was adopted to analyze the immunoreactivity of walnut allergens treated with HHP and thermal, which indicated that HHP has great potentiality for industrialization to eliminate or decrease allergenicity of food protein.

Mass spectrometric analysis of allergens in roasted walnuts

Thermal processing of allergenic foods can induce changes in the foods' constituent allergens, but the effects of heat treatment are poorly defined. Like other commonly allergenic tree nuts, walnuts often undergo heat treatment (e.g. roasting or baking) prior to consumption. This study evaluated the changes in solubility and detectability of allergens from roasted walnuts using tandem mass spectrometry methods. Walnuts were roasted (132°C or 180°C for 5, 10, or 20min) and prepared for LC-MS/MS using sequential or simultaneous extraction and tryptic digestion protocols. The LC-MS/MS data analysis incorporated label-free quantification of relevant allergens and Maillard adduct screening. In some proteins (2S albumin, LTP, and the 7S globulin N-terminal region) minor changes in relative abundance were observed following roasting. The mature 7S and 11S globulins, however, showed significantly increased detection following roasting at 180°C for 20min when using the simultaneous extraction/digestion protocol, possibly due to increased digestibility of the proteins. The results of this study indicate that individual walnut allergens respond differently to thermal processing, and the detection of these proteins by LC-MS/MS is dependent on the protein in question, its susceptibility to proteolytic digestion, the degree of thermal processing, and the sample preparation methodology. SignificanceUnderstanding the behavior of food allergens in the context of relevant food matrices is critical for both food allergen management and for elucidating matrix and processing-associated factors influencing protein allergenicity. The use of mass spectrometry to identify food allergens and detect allergenic food residues has been increasingly developed due to the advantages associated with the direct, sequence-level analysis possible with MS. To date, however, few studies have implemented MS technology to analyze the effects of thermal processing on allergenic food proteins. The MS analysis results presented in this study revealed not only information about the molecular-level effects of roasting on walnut allergens but also data pertinent to the development of MS-based detection methods for walnut residues in food products.

Detection by real time PCR of walnut allergen coding sequences in processed foods

A quantitative real-time PCR (RT-PCR) method, employing novel primer sets designed on Jug r 1, Jug r 3, and Jug r 4 allergen-coding sequences, was set up and validated. Its specificity, sensitivity, and applicability were evaluated. The DNA extraction method based on CTAB–phenol–chloroform was best for walnut. RT-PCR allowed a specific and accurate amplification of allergen sequence, and the limit of detection was 2.5pg of walnut DNA. The method sensitivity and robustness were confirmed with spiked samples, and Jug r 3 primers detected up to 100mg/kg of raw walnut (LOD 0.01%, LOQ 0.05%). Thermal treatment combined with pressure (autoclaving) reduced yield and amplification (integrity and quality) of walnut DNA. High hydrostatic pressure (HHP) did not produce any effect on the walnut DNA amplification. This RT-PCR method showed greater sensitivity and reliability in the detection of walnut traces in commercial foodstuffs compared with ELISA assays.

Insoluble and soluble roasted walnut proteins retain antibody reactivity

Thermal processing techniques commonly used during food production have the potential to impact food allergens by inducing physical and/or chemical changes to the proteins. English walnuts (Juglans regia) are among the most commonly allergenic tree nuts, but little information is available regarding how walnut allergens respond to thermal processing. This study evaluated the effects of dry roasting (132 or 180°C for 5, 10, or 20min) on the solubility and immunoreactivity of walnut proteins. A dramatic decrease in walnut protein solubility was observed following dry roasting at 180°C for 20min. However, both the soluble and insoluble protein fractions from roasted walnuts maintained substantial amounts of IgG immunoreactivity (using anti-raw and anti-roasted walnut antisera), with similar patterns of reactivity observed for human IgE from walnut-allergic individuals. Thus, walnut proteins are relatively stable under certain thermal processing conditions, and IgE reactivity remains present even when insoluble aggregates are formed.

Microarrayed Jug r 1 shows the best diagnostic performance in walnut allergy

Several walnut (WN) allergens have been identified to date: Jug r 1 (2S albumin), Jug r 2 (7S globulin), Jug r 3 (LTP), Jug r 4 (11S globulin) and Jug r 5 (profilin), but their clinical significance is still to be determined in some cases. Allergen microarray (ISAC) provides information for three of these allergens. The aim of the study was to determine the importance of component resolved diagnosis in WN allergic patients. Ninety children suspected to have allergy to fruit, nuts and/or legumes were selected. Patients were classified as allergic if they presented at least 2 reactions unequivocally related to WN ingestion in the last 2 years. Patients were defined as tolerant if they consumed WN on a regular basis. Clinical questionnaire, skin prick test (SPT), total and specific IgE and ImmunoCAP ISAC (Thermo Fisher) were performed. Thirty-one patients (18 males) were defined as allergic and 59 (36 males) tolerant. WN-SPT wheal size (median diameter 5.25 mm; IQR: 4-7 vs. 0 mm; IQR: 0-2, p=0.000) and WN-sIgE (median 5.35 kU/L; IQR: 3.52-12.84 vs. 0.65 kU/L; IQR: 0.08-1.86, p=0.000) were significantly greater in allergic than in tolerant children. Positive Jug r 1-sIgE was significantly more frequent in allergic patients (80.65% vs. 5.08%, p=0.000). Jug r 1-sIgE values were also significantly higher in allergic children (median 2.5 ISU; IQR: 0-7.6 vs. 0 ISU; IQR: 0-0, p=0.000). This was not found for Jug r 2 or Jug r 3. ROC curves were constructed for the three allergens showing Jug r 1 the best diagnostic performance (AUC: 0.876, 95%CI: 0.788-0.965, p=0.000) (Figure ​(Figure11 and Table ​Table11). Figure 1 ROC curves for Walnut allergens Table 1 Diagnostic performance of walnut allergens (MAISAC) In conclusion, Jug r 1-sIgE is the best discriminating allergen in WN allergic patients.

Characterization of low molecular weight allergens from English walnut (Juglans regia).

Although English walnut is a commonly allergenic tree nut, walnut allergens have been poorly characterized to date. The objective of this work was to characterize the natural, low molecular weight (LMW) allergens from walnut. A protocol was developed to purify LMW allergens (specifically 2S albumins) from English walnuts. In addition to 2S albumins, a series of peptides from the N-terminal region of the 7S seed storage globulin proprotein were also identified and characterized. These peptides comprised a four-cysteine motif (C-X-X-X-C-X10-12-C-X-X-X-C) repeated throughout the 7S N-terminal region. Upon IgE immunoblotting, 3/11 and 5/11 sera from walnut-allergic subjects showed IgE reactivity to the 7S N-terminal fragments and 2S albumin, respectively. The mature 7S protein and the newly described 7S N-terminal peptides represent two distinct types of allergens. Because the proteolytic processing of 7S globulins has not been elucidated in many edible plant species, similar protein fragments may be present in other nuts and seeds.

Walnut allergens: molecular characterization, detection and clinical relevance

Food-induced allergies have been regarded as an emergent problem of public health. Classified as important allergenic ingredients, the presence of walnut and other nuts as hidden allergens in processed foods constitutes a risk for sensitized individuals, being a real problem of allergen management. Attending to the increasing importance dedicated to walnut allergy, this review intends to provide the relevant and up-to-date information on main issues such as the prevalence of walnut allergy, the clinical threshold levels, the molecular characterization of walnut allergens and their clinical relevance, as well as the methodologies for walnut allergen detection in foods. As the walnut used in human diet comes from Juglans regia and Juglans nigra, the molecular characterization of the allergens from both species included in the prolamins (Jug r 1, Jug n 1 and Jug r 3), cupins (Jug r 2, Jug n 2 and Jug r 4) and profilins (Jug r 5), together with respective clinical relevance, were compiled in this review. The most recent progresses on walnut allergen detection techniques (protein- and DNA-based) are described and critically compared, including the emergent multitarget approaches.

Development of an indirect competitive immunoassay for walnut protein component in food

Among food allergens, walnut is a frequent cause of adverse food reactions in allergic patients. In this study, the walnut allergen protein 2S albumin precursor (Jug r 1) cDNA was synthesised and cloned into the pGEX-6P-1 expression vector. The recombinant plasmids were transformed into Escherichia coli (E. coli) BL21(DE3) pLys for expression of protein Jug r 1. Polyclonal antibodies were prepared against the expressed purified Jug r 1 protein. An indirect competitive enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of walnut soluble proteins in processed foods was developed using the prepared polyclonal antibodies. The developed ELISA had a high specificity, walnut protein standard solution at 2.2ng/mL [inhibition concentration (IC80) of the competitive test] was clearly identified by the ELISA. The mean recoveries ranged from 86% to 112%. The coefficient of variation (CV) for the 4 model foods was 6.4–8.7%.

Effect of thermal processing on the performance of the novel single-tube nested real-time PCR for the detection of walnut allergens in sponge cakes

Walnut is commonly included as an ingredient in bakery products, being widely appreciated by many consumers. However, walnut is also classified as an allergenic food, representing a potential risk for the health of sensitised individuals when present in processed foods as a hidden allergen. In the present work, we developed a single-tube nested real-time PCR system to trace walnut in bakery products and assessed the effect of heat treatment on the performance of the technique. The proposed approach, targeting the sequence encoding for the Jug r 3 allergen, enabled lowering the relative limit of detection (LOD) of the conventional real-time PCR assay from 0.005% to 0.001% of walnut in both batter and sponge cakes. The absolute LOD was 1pg of walnut DNA (1.6 DNA copies) in both mixtures, evidencing that the performance of the method was not affected by the heat processing. To our knowledge we proposed for the first time a single-tube nested real-time PCR system as a cost-effective, robust and powerful tool for high-throughput DNA-based detection and quantification of walnut allergens in raw and processed foods.