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 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.     

Quantitative sandwich ELISA for the determination of lupine (Lupinus spp.) in foods

The use of lupine in foods has increased considerably during the past decade, reflected by a corresponding increase in reported lupine-induced allergic incidents. Lupine allergy may arise either by primary sensitization or by clinical cross-reactivity in peanut-allergic persons. Detection of lupine proteins in food has previously been based on the use of patient serum. A novel sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of lupine in processed foods was developed, using a polyclonal rabbit antilupine capture antibody and a biotinylated conjugate of the same antibody for detection. The antibody was highly specific for lupine, apart from minor cross-reactivities to other legumes. The assay had a detection limit of 1 mug/g and was successfully used to quantify lupine protein in various food matrixes. Recoveries ranged from 60 to 116%, while the intra-and interassay coefficients of variation were <6% and <21%, respectively.     

Novel polyclonal-monoclonal-based ELISA utilized to examine lupine (Lupinus species) content in food products

Sweet lupines are increasingly used in food production. Cause for concern has been expressed due to the increase in reported lupine-induced allergic incidents and the association between lupine and peanut allergies. In the current study, a polyclonal-monoclonal antibody-based sandwich ELISA for the detection of lupine proteins in foods was developed. The assay was sensitive to both native and processed proteins from Lupinus angustifolius and Lupinus albus and had a detection limit of 1 mug/g. Intra- and interassay coefficients of variation were <5 and <17%, respectively. A selection of 112 food samples, both with and without lupine declaration, was evaluated for their content of lupine. The data showed that the majority were in agreement with the respective labeling. However, some inconsistency was seen, typically in bread/rolls and soy flours.     

Confirmation of the allergenic peanut protein, Ara h 1, in a model food matrix using liquid chromatography/tandem mass spectrometry (LC/MS/MS)

Enzymatic digestion of total protein along with liquid chromatography/tandem mass spectrometry (LC/MS/MS) was used to confirm the presence of a major peanut allergen in food. Several peptides obtained from the enzymatic digestion of the most abundant peanut allergen, Ara h 1, were identified as specific peptide biomarkers for peanut protein. Using ice cream as a model food matrix, a method was developed for the detection of the allergen peptide biomarkers. A key component of the method was the use of molecular mass cutoff filters to enrich the Ara h 1 in the protein extracts. By applying the method to ice cream samples containing various levels of peanut protein, levels as low as 10 mg/kg of Ara h 1 could routinely be detected. This method provides an unambiguous means of confirming the presence of the peanut allergen, Ara h 1, in foods and can easily be modified to detect other food allergens.     

Effect of processing on recovery and variability associated with immunochemical analytical methods for multiple allergens in a single matrix: sugar cookies

Among the major food allergies, peanut, egg, and milk are the most common. The immunochemical detection of food allergens depends on various factors, such as the food matrix and processing method, which can affect allergen conformation and extractability. This study aimed to (1) develop matrix-specific incurred reference materials for allergen testing, (2) determine whether multiple allergens in the same model food can be simultaneously detected, and (3) establish the effect of processing on reference material stability and allergen detection. Defatted peanut flour, whole egg powder, and spray-dried milk were added to cookie dough at seven incurred levels before baking. Allergens were measured using five commercial enzyme-linked immunosorbent assay (ELISA) kits. All kits showed decreased recovery of all allergens after baking. Analytical coefficients of variation for most kits increased with baking time, but decreased with incurred allergen level. Thus, food processing negatively affects the recovery and variability of peanut, egg, and milk detection in a sugar cookie matrix when using immunochemical methods.     

Protein quality, antigenicity, and antioxidant activity of soy-based foodstuffs

Commercial soy-based foodstuffs, including beverages ( n = 15), cow's milk supplemented with soy isoflavones ( n = 1), snacks ( n = 1), and biscuits ( n = 2), were analyzed to find any link between alterations in protein quality, safety (antigenicity), functionality (antioxidant activity), and food processing. Protein content was analyzed by the Kjeldhal method and available lysine by OPA assay. Chromatographic (RP-HPLC) and electrophoretic (SDS-PAGE) protein profiles were obtained to monitor modifications in the structure of soy allergens. The antigenicity was estimated by immunoblotting against soy total antibodies. Total phenol content was measured by Folin-Ciocalteu, while peroxyl radical scavenging activity of the sample was determined by ORAC FL assay. Protein content did not differ of those declared by the producers. Lysine availability was higher in liquid soy beverages compared to that in other soy foodstuffs studied here. 7S and 11S soy allergens were detected by RP-HPLC and SDS-PAGE, respectively. Both data indicated changes in soy protein patterns due to processing of instant powdered soymilk, soy snacks, and biscuits. Immunoblotting assay showed modifications in the antigenic response of these foodstuffs based on soy, suggesting that their processing had altered the structure of soy allergens. RP-HPLC, SDS-PAGE, and immunoblotting resulted in adequate analytical approaches for detecting changes in protein structure due to processing and adulteration. Protein quality, antigenicity, and antioxidant activity of soy products can be affected as a function of the intensity of the thermal processing.     

A reliable and sensitive immunoassay for the determination of crustacean protein in processed foods

Among food allergens, crustacea such as shrimps, crabs, and lobsters are a frequent cause of adverse food reactions in allergic patients. The major allergen has been identified as a muscular protein, tropomyosin. A novel sandwich enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of crustacean protein in processed foods was developed using the sample dilution buffer that is added to porcine tropomyosin. The sandwich ELISA method was highly specific for the Decapoda group, apart from minor cross-reactivities to other crustacea and mollusks. The recovery ranged from 85 to 141%, while the intra- and interassay coefficients of variation were less than 2.8 and 8.4%, respectively.     

Confirmation of peanut protein using peptide markers in dark chocolate using liquid chromatography-tandem mass spectrometry (LC-MS/MS)

Detection of peptides from the peanut allergen Ara h 1 by liquid chromatography-mass spectrometry (LC-MS) was used to identify and estimate total peanut protein levels in dark chocolate. A comparison of enzymatic digestion subsequent to and following extraction of Ara h 1 from the food matrix revealed better limits of detection (LOD) for the pre-extraction digestion (20 ppm) than for the postextraction digestion (50 ppm). Evaluation of LC-MS instruments and scan modes showed the LOD could be further reduced to 10 ppm via a triple-quadrupole and multiple-reaction monitoring. Improvements in extraction techniques combined with an increase in the amount of chocolate extracted (1 g) improved the LOD to 2 ppm of peanut protein. This method provides an unambiguous means of confirming the presence of the peanut protein in foods using peptide markers from a major allergen, Ara h 1, and can easily be modified to detect other food allergens.     

Immunodiffusion screening method for detection of motile Salmonella in foods: collaborative study

A collaborative study was performed to validate the performance of the 1-2 TEST for detection of motile salmonellae in foods. Detection is based on observation of an immobilized band of cells. Twenty-three laboratories participated in the study. The 1-2 TEST (immunodiffusion test) was compared with the standard culture procedure (BAM/AOAC; FDA Bacteriological Analytical Manual) for detection of Salmonella in 6 food types: ground black pepper, soy flour, dried whole egg, milk chocolate, nonfat dry milk, and raw deboned turkey. Uninoculated and inoculated samples were included in each food group analyzed. After the tests on the 6 foods were completed, analysis of the data for turkey and soy flour showed that certain collaborators obtained data inconsistent with the data from the majority of collaborators. No specific method deviations to account for the inconsistencies were reported by those collaborators, so the collaborative testing of these 2 foods was repeated. Analysis of data for pepper, chocolate, nonfat dry milk, dried whole egg, and the second set of soy flour and turkey indicated 96.1% agreement between the BAM/AOAC and immunodiffusion test methods. The false negative rates for the immunodiffusion test and BAM/AOAC methods were 3.6 and 1.7%, respectively. There was no significant difference in the productivity of the immunodiffusion test and BAM/AOAC method at the 5% level for any of the 6 foods. The immunodiffusion screening method has been approved official first action for detection of motile Salmonella in foods.     

Immunoassay to quantify the major peach allergen Pru p 3 in foodstuffs. Differential allergen release and stability under physiological conditions

Pru p 3 is a lipid transfer protein (LTP) that has been identified as the major peach (Prunus persica) allergen. However, little is known about the amount present in both raw and processed foodstuffs. Moreover, the in vivo release upon consumption of peach-containing foods remains unclear. We have developed a sensitive monoclonal antibody-based ELISA for Pru p 3. The method has been applied to measure the allergen levels in foodstuffs and the allergen release under different physiological conditions. A significant variability in all raw peaches and peach-containing foods tested has been detected. The allergen was extracted more efficiently at a low pH, and it was highly resistant to pepsin. This ELISA will be very useful in controlling the allergen concentration in diagnostics, in evaluating threshold levels in provocation tests, and in detecting hidden allergens in processed foods and cosmetics.     

Establishment of selenium uptake and species distribution in lupine, Indian mustard, and sunflower plants

Selenium has been recognized as essential for all mammals; therefore, its concentration level and speciation are of great concern. Plants are one of the main sources of selenium in the diet. Thus, inorganic selenium uptake and its transformation in different species were evaluated in Indian mustard (Brassica juncea), sunflower (Helianthus annus), and white lupine (Lupinus albus). More than 1.2 g x kg(-)(1) (dry matter) of Se was found in the aerial part of Indian mustard when growing on 1 mg x L(-)(1) of Se as Na(2)SeO(4), and approximately half this amount was determined in the leaves of the lupine, which is still quite high. Selenomethionine was the main selenium-containing amino acid identified in most of the extracts by HPLC-ICP-MS. The higher values were 6.8 and 14.5 mg x kg(-)(1) (expressed as Se in dry matter) in the leaves of lupine and sunflower, respectively. This is of great importance because some authors have considered the combination of this enriched material with non-enriched food as a source of selenium supplementation.     

Development of a real-time PCR for the detection of lupine DNA (lupinus species) in foods

Lupine flour, protein, and fiber have become common ingredients in food products. The association of lupine-related allergic incidents with peanut allergy is a cause for concern as the latter may bring about severe reactions. In this study, a hybridization probe-based real-time PCR assay for the detection of lupine DNA in foods was developed. Particular attention was paid to the specificity of the method, which was verified by analysis of DNA extracts from more than 50 potential food ingredients such as legumes, cereals, seeds, nuts, spices, fruits, and meat. The limit of detection of the method was determined as 0.1 mg/kg. The successful detection of the presence/absence of lupine DNA in 20 samples proved the suitability of the assay for the analysis of frequently encountered food matrices.     

Real-time polymerase chain reaction (PCR) quantitative detection of Brassica napus using a locked nucleic acid TaqMan probe

Several countries have introduced mandatory labeling requirements on foods derived from genetically modified organisms. Real-time quantitative Polymerase Chain Reaction (PCR) has quickly become the method of choice in support of these regulations and requires the development of separate PCR assays targeting the transgenic sequence as well as a specific endogenous gene sequence. To develop a Brassica napus-specific PCR assay, partial sequences of the acetyl-CoA carboxylase BnACCg8 gene from B. napus and the closely related Brassica rapa were determined and compared, and a region of unique nucleotide sequence was identified. Universal amplification primers were designed to either side of this region, and a locked nucleic acid TaqMan probe was designed to the B. napus-specific sequence. Evaluation of this primer/probe combination indicated a high level of specificity to B. napus: no amplification signal was observed with any other species tested, including five closely related Brassica species. The method was assayed with 14 different B. napus cultivars, and comparable amplification curves were consistently obtained for all. The assay was highly sensitive, with a limit of detection between 1 and 10 haploid copies. Practically, the method was demonstrated to be effective for the detection of processed food samples and for the quantification of Roundup Ready canola content in mixed samples.     

Marzipan: polymerase chain reaction-driven methods for authenticity control

According to German food guidelines, almonds are the only oilseed ingredient allowed for the production of marzipan. Persipan is a marzipan surrogate in which the almonds are replaced by apricot or peach kernels. Cross-contamination of marzipan products with persipan may occur if both products are produced using the same production line. Adulterations or dilutions, respectively, of marzipan with other plant-derived products, for example, lupine or pea, have also been found. Almond and apricot plants are closely related. Consequently, classical analytical methods for the identification/differentiation often fail or are not sensitive enough to quantify apricot concentrations below 1%. Polymerase chain reaction (PCR)-based methods have been shown to enable the differentiation of closely related plant species in the past. These methods are characterized by high specificity and low detection limits. Isolation methods were developed and evaluated especially with respect to the matrix marzipan in terms of yield, purity, integrity, and amplificability of the isolated DNA. For the reliable detection of apricot, peach, pea, bean, lupine, soy, cashew, pistachio, and chickpea, qualitative standard and duplex PCR methods were developed and established. The applicability of these methods was tested by cross-reaction studies and analysis of spiked raw pastes. Contaminations at the level of 0.1% could be detected.     

Fast real-time PCR for the detection of crustacean allergen in foods

Crustaceans are one of the most common allergens causing severe food reaction. These food allergens are a health problem, and they have become very important; there are various regulations that establish that labeling must be present regarding these allergens to warn consumers. In the present work a fast real-time PCR, by a LNA probe, was developed. This allows the detection of crustaceans in all kinds of products, including processed products in which very aggressive treatments of temperature and pressure during the manufacturing process are used. This methodology provides greater sensitivity and specificity and reduces the analysis time of real-time PCR to 40 min. This methodology was further validated by means of simulating products likely to contain this allergen. For this, products present on the market were spiked with crustacean cooking water. The assay is a potential tool in issues related to the labeling of products and food security to protect the allergic consumer.     

Detecting potential IgE-reactive sites on food proteins using a sequence and structure database, SDAP-food

The high incidence of food allergies, including oral allergy syndrome, represent major considerations when introducing new crops and foods. A new structural database of allergenic proteins, SDAP-Food, http://fermi.utmb.edu/SDAP/, has been developed to aid in predicting the IgE-binding potential of novel food proteins and cross-reactivities among known allergens. The site is designed to facilitate the first steps of a decision tree approach to determine the allergenicity of a given protein, based on the sequence and structural similarity to known allergens and their IgE binding sites. Immunological tests can then be used to confirm the predictions. A hierarchical procedure for identifying potential allergens, using a physical property-based sequence similarity index, has been designed to identify regions that resemble known IgE binding sites. As an example, SDAP tools were used to find food allergen sequences similar to an IgE binding site of the Jun a 3 allergen from mountain cedar pollen. The SDAP sequence similarity search matched the Jun a 3 epitope to regions in several food allergens, including cherry (Pru av 2), apple (Mal d 2) and pepper (Cap a 1), which are, like Jun a 3, members of the plant pathogenesis-related (PR-5) protein family. Homology modeling, using our EXDIS/DIAMOD/FANTOM program suite, indicated a similar surface location and structure for the potential epitope region on all of these allergens. The quantitative approach presented here can be used as part of a screening process for potential allergenicity of recombinant food products.     

Validation of a rice specific gene, sucrose phosphate synthase, used as the endogenous reference gene for qualitative and real-time quantitative PCR detection of transgenes

With the development of transgenic crops, many countries have issued regulations to label the genetically modified organisms (GMOs) and their derived products. Polymerase Chain Reaction (PCR) methods are thought to be reliable and useful techniques for qualitative and quantitative detection of GMOs. These methods generally need to amplify the transgene and compare the amplified result with that of the corresponding reference gene to obtain reliable results. In this article, we reported the development of specific primers and probe for the rice (Oryza sativa) sucrose phosphate synthase (SPS) gene and PCR cycling conditions suitable for the use of this sequence as an endogenous reference gene in both qualitative and quantitative PCR assays. Both methods were assayed with 13 different rice varieties, and identical amplification products were obtained with all of them. No amplification products were observed when DNA samples from other species, such as wheat, maize, barley, tobacco, soybean, rapeseed, tomato, sunflower, carrot, pepper, eggplant, lupine, mung bean, plum, and Arabidopsis thaliana, were used as templates, which demonstrated that this system was specific for rice. In addition, the results of the Southern blot analysis confirmed that the SPS gene was a single copy in the tested rice varieties. In qualitative and quantitative PCR analyses, the detection sensitivities were 0.05 and 0.005 ng of rice genomic DNA, respectively. To test the practical use of this SPS gene as an endogenous reference gene, we have also quantified the beta-glucuronidase (GUS) gene in transgenic rice using this reference gene. These results indicated that the SPS gene was species specific, had one copy number, and had a low heterogeneity among the tested cultivars. Therefore, this gene could be used as an endogenous reference gene of rice and the optimized PCR systems could be used for practical qualitative and quantitative detection of transgenic rice.     

Allergenicity assessment of osmotin, a pathogenesis-related protein, used for transgenic crops

Genetic engineering can enhance abiotic stress tolerance of plants, thereby increasing productivity. The present study investigates allergenicity of osmotin protein used for developing transgenic crops. Bioinformatic analysis of osmotin was performed using SDAP and Farrp allergen databases. Osmotin was cloned in pET22b+ vector, purified to homogeneity, and analyzed for digestibility, heat stability, and IgE binding using atopic patients' sera. Osmotin showed 40-92% and 48-75% homology with allergens in SDAP and Farrp databases, respectively. These cross-reactive allergens were from apple, tomato, peach, capsicum, kiwi fruit, and cypress. Osmotin was resistant to pepsin digestion and heat treatment at 90 °C for 1 h. Osmotin protein showed dose-dependent inhibition with pooled patients' sera. It showed significant IgE binding with 22 of 117 patients' sera who were sensitized to tomato and apple, thus indicating cross-reactivity among tomato, apple, and osmotin allergens. In conclusion, osmotin was identified as a potential allergen and showed cross-reactivity with tomato and apple allergens.     

Colorimetric deoxyribonucleic acid hybridization assay for rapid screening of Salmonella in foods: collaborative study

A collaborative study was performed in 11 laboratories to validate a colorimetric DNA hybridization (DNAH) method for rapid detection of Salmonella in foods. The method was compared to the standard culture method for detection of Salmonella in nonfat dry milk, milk chocolate, soy isolate, dried whole egg, ground black pepper, and raw ground turkey. Samples inoculated with high (0.4-2 cells/g) and low (0.04-0.2 cells/g) levels of Salmonella and uninoculated control samples were included in each food group analyzed. There was no significant difference in the proportion of samples positive by DNAH and culture procedure for any of the 6 foods. The colorimetric DNA hybridization assay screening method has been adopted official first action as a rapid screening method for detection of Salmonella in all foods.