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.     

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.     

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.     

Flower analysis as a new approach to diagnosing the nutritional status of the peach tree

The chemical diagnosis of the nutritional status of peach (Prunus persica L. Batsch) trees by leaf analysis is done too late to allow any deficiencies to be corrected, especially with early bearing varieties. The use of flower analysis would be it possible to detect and treat any deficiencies at an earlier stage. Based on the relationships found between nutrient concentration of flowers at full bloom and in leaves taken 60 and 120 days after full bloom (DAFB), we have established the following reference values for the interpretation of floral analyses of this crop: N = 2.91±0.27; P = 0.39±0.05; K = 1.70±0.26; Ca = 0.87±0.16; and Mg = 0.23±0.02 expressed as a percentage of the dry matter weight of flowers.     

A novel approach for the detection of potentially hazardous pepsin stable hazelnut proteins as contaminants in chocolate-based food

Contamination of food products with pepsin resistant allergens is generally believed to be a serious threat to patients with severe food allergy. A sandwich type enzyme-linked immunosorbent assay (ELISA) was developed to measure pepsin resistant hazelnut protein in food products. Capturing and detecting rabbit antibodies were raised against pepsin-digested hazelnut and untreated hazelnut protein, respectively. The assay showed a detection limit of 0.7 ng/mL hazelnut protein or <1 microg hazelnut in 1 g food matrix and a maximum of 0.034% cross-reactivity (peanut). Chocolate samples spiked with 0.5-100 microg hazelnut/g chocolate showed a mean recovery of 97.3%. In 9/12 food products labeled "may contain nuts", hazelnut was detected between 1.2 and 417 microg hazelnut/g food. It can be concluded that the application of antibodies directed to pepsin-digested food extracts in ELISA can facilitate specific detection of stable proteins that have the highest potential of inducing severe food anaphylaxis.     

Phenotype management: a new approach to habitat restoration

The goal of habitat restoration is to provide environmental conditions that promote the maintenance and growth of target populations. But rarely is it considered how the allocation of resources influences the diversity of phenotypes in these populations. Here we present a framework for considering how habitat restoration can shape the development and expression of phenotypes. We call this approach phenotype management as it entails restoring the resources in a habitat to manage phenotypic diversity. Phenotype management is achieved by manipulating the spatial and temporal distribution of resources to alter the degree of competition among individuals. Differences in competition, in turn, lead to changes in phenotypic and life history expression that affect population parameters including demography and effective population size (N_e). To illustrate how phenotype management can be applied, we explore how resource distributions shape variation in phenotypes in two imperiled fishes, Pacific salmon and desert pupfish. In both examples, modulating male reproductive phenotypes changes the allocation of reproductive success among population members to subsequently affect N_e. These examples further demonstrate that whether to increase or decrease phenotypic diversity depends on the primary conservation pressures faced by the species.     

Development of a real-time PCR method to detect potentially allergenic sesame (Sesamum indicum) in food

Recent papers indicate that the prevalence of allergic reactions to sesame (Sesamum indicum) is increasing in European countries. This paper describes the development of a selective real-time PCR method for the detection of sesame in food. The assay did not show any cross-reactivity with 17 common food ingredients. The real-time PCR method was applied to determine sesame in several crackers, salty snacks, biscuits, tahina sesame paste and sesame oil. With the exception of sesame oil, in all of the samples where sesame was declared, sesame was detected by the real-time PCR assay (Ct value<35). In the samples which might contain sesame or where sesame was not listed, sesame could not be detected (Ct value>35).     

Comparison of real-time PCR detection chemistries and cycling modes using Mon810 event-specific assays as model

The most widely accepted methods for accurate quantitative detection of genetically modified organisms rely on real-time PCR. Various detection chemistries are available for real-time PCR. They include sequence-unspecific DNA labeling dyes such SYBR-Green I and the use of both universal (e.g., AmpliFluor) and sequence-specific double-labeled probes, the latter comprising hybridization (e.g., Molecular Beacon) and hydrolysis (e.g., TaqMan or MGB) probes. Also, new real-time PCR devices and reagents allowing fast cycling reactions exist. Five Mon810 real-time PCR assays were developed in which the event specificity was based on the detection of transgene and plant rearranged sequences found to 3' flank the insertion site. Every assay was specifically designed for one particular detection chemistry, that is, AmpliFluor, Molecular Beacon, MGB, TaqMan, and SYBR-Green I. When possible, the assays were adapted to fast cycling mode. All assays displayed satisfactory performance parameters, although Molecular Beacon, MGB, and TaqMan chemistries were the most suitable for quantification purposes in both conventional and fast cycling modes.     

A new approach to elaborate a multifunctional soil quality index

The formulation of quality indices for a given land area can be considered of great practical interest, both for highlighting the specific characteristics of that area, and for establishing the best management methods to adopt in order to exploit the soil’s maximum potential. Among other things, the specific formulation of the parameters considered here enables us to simulate specific measures designed to modify poor chemical, physical or biological characteristics (AQI, MQI), or to achieve a more rational form of soil management (AQI). We believe that one of the most interesting aspects of the proposed method is its tree-structured approach, which enables us to formulate an overall synthetic index by going through successive levels of aggregation. In this way, we are able to meet the contrasting needs of those, on the one hand, who are concerned about the inevitable loss of information inherent in every process of synthesis, and those, on the other hand, whose decision-making tasks require them to have concise quantitative information. The choice concerning which level of aggregation to operate will strictly depend on the objectives one is pursuing through the use of such indices.     

Soil and water degradation processes in burned areas: Lessons learned from a nested approach

Forest fires produce a major impact on soil, water and vegetation. Despite the amount of research published on this subject, there are two major problems that hamper the fully understanding of on and off-site impacts of forest fires. They include methodological problems steaming from the uniqueness of burned soil properties, easily erodible, by the fast degradation they undergo over a short period of time immediately after fire and by the meaning of the impacts at different scales.     

Development of a real-time PCR method to quantify the PGPR strain Azospirillum lipoferum CRT1 on maize seedlings

Azospirillum lipoferum CRT1 is a promising phytostimulatory PGPR for maize, whose effect on the plant is cell density-dependent. A nested PCR method is available for detection of the strain but does not allow quantification. The objective was to develop a real-time PCR method for quantification of A. lipoferum CRT1 in the rhizosphere of maize seedlings. Primers were designed based on a strain-specific RFLP marker, and their specificity was verified under qualitative and quantitative PCR conditions based on successful CRT1 amplification and absence of cross-reaction with genomic DNA from various rhizosphere strains. Real-time PCR conditions were then optimized using DNA from inoculated or non-inoculated maize rhizosphere samples. The detection limit was 60 fg DNA (corresponding to 19 cells) with pure cultures and 4 × 10⁴ CFU equivalents g⁻¹ lyophilized sample consisting of mixture of rhizosphere soil and roots. Inoculant quantification was effective down to 10⁴ CFU equivalents g⁻¹. Assessment of CRT1 rhizosphere levels in a field trial was in accordance with estimates from semi-quantitative PCR targeting another locus. This real-time PCR method, which is now available for direct rhizosphere monitoring of A. lipoferum CRT1 in greenhouse and field experiments, could be used as a reference for developing quantification tools for other Azospirillum inoculants.     

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.     

Establishment of a highly sensitive sandwich enzyme-linked immunosorbent assay specific for ovomucoid from hen's egg white

A highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) kit was established for quantifying ovomucoid from hen's egg white, which has been considered as one of the major allergen in egg white. The detection limit reached 0.041 ng/mL, and linearity ranged from 0.1 to 6.25 ng/mL. Intra- and interassay coefficient variations were all lower than 5% at three concentrations (0.5, 2.5, and 5 ng/mL). No cross-reactivity was observed with bovine serum, horse serum, goat serum, human serum, duck egg white, goose egg white, quail egg white, and pigeon egg white, but a low level of cross-reactivity was found with chicken serum. The ELISA kit was established on the basis of two monoclonal antibodies (mAbs) recognizing different epitopes of ovomucoid. However, these mAbs were generated using commercially purified ovalbumin as immunogen. Studies on the relative allergenicity and antigenicity of egg white protein have been performed by many researchers, but there were controversial opinions reported previously because of the impurity of each egg white protein used in various studies. In the present work we measured the degree of ovomucoid contamination in commercially purified ovalbumin sample, and the value was about 11%. We also determined the ovomucoid residue in influenza vaccine samples for the first time. These data showed that the ELISA kit we established could serve as an effective method for precisely quantifying concentrations of ovomucoid in the egg industry and as a useful tool for the research of allergenicity and antigenicity of hen's egg proteins.     

A methodological approach to estimate the geogenic contribution in soils potentially polluted by trace elements. Application to a case study

Purpose

The determination of the contribution of background values in a potentially polluted soil is very important in defining the contamination extension, in particular in areas of geological complexity and long-term economic development, where mining and industry have been traditional activities and soils are showing both geogenic and anthropogenic contributions. Some approaches have been proposed for the estimation of the anthropogenic input vs. the background; in this paper we present a more robust approach.

Materials and methods

The proposed methodological approach includes the following steps. The first step consists of the comparison among the trace element contents in potentially polluted soils (PPS) and the reference and threshold values calculated both for the same geotectonic unit. A second stage is the calculation of the reference and threshold values for the surrounding area (LTV), natural setting, of the PPS with similar lithological characteristics. The final step is based on the analysis of the results by comparison of the PPS with LTV. On the other hand, the definition of a new pollution factor allows to grade the pollution and to classify the pollution importance.

Results and discussion

The protocol proposed was applied to PPS from a potentially polluted area of SW Spain. The anthropogenic vs. geogenic anomalies and the pollution grade of the three PPS were assessed, which is important to establish the priority to further actions. In addition, this study makes clear that the use of the enrichment factors to estimate the pollution of soils is not advisable. On the other hand, in this study, new areas close to the PPS were defined as potentially polluted because of the high trace element concentration.

Conclusions

The methodological approach proposed can be considered as a good indicator for evaluating the geogenic vs. anthropogenic contribution in polluted soils and for classifying the pollution importance in a more robust way than the use of other previous indexes. The proposal methodology could be used also by the administration to detect other PPS in a study area, which a priori were not considered as contaminated.     

The development of a new approach for establishing a core collection using multivariate analyses with tulip as case

A methodology is developed for the establishment of a core collection based on phenotypic data. A case is worked out for the tulip cultivar collection of the Hortus Bulborum, consisting of approximately 1000 cultivars. The methodology is based on cluster analysis of phenetic characters, selection of cultivars from the resulting clusters according to a combination of the proportional and genetic diversity dependent strategies, optimisation of the selection, and validation by means of principal component analysis and by the diversity indices of Nei and Shannon & Weaver. A core collection with 104 cultivars (approx. 10%: CORE-1) did not give a sufficient representation of the existing diversity. A set of 152 cultivars (approx. 15%: CORE-2) showed a far better representation. The variation in resistance levels for Fusarium is almost completely represented in CORE-2. Although a set of 200 cultivars (approx. 20%: CORE-3) reached a nearly complete representation of the total diversity, a better representation of the resistance levels was not achieved. The newly presented methodology for defining a core collection appears to be a useful approach. The included steps for optimising and validating the choice of accessions makes it a reliable and broadly applicable method.     

A new approach to determining water stable aggregation

Abstract

A new method is introduced to measure water stability of soil aggregates. The wrist‐action shaker is a simple, inexpensive tool that provides highly accurate data for the assessment of soil erodibility. Three soils from Hawaii (two Oxisols and one Vertisol) with different mineralogies, management histories, and potassium (K)‐factors were examined in this study. Six indices of water stable aggregation were determined after rapid immersion of air‐dry aggregates, followed by gentle wet‐sieving. Single‐sieve indices of percent water stable aggregates (WSA) < 0.063 mm, > 0.25 mm, and > 1.00 mm, were highly correlated. Additionally, these indices were highly correlated with three multiple sieve indices, namely geometric mean aggregate diameter (GMAD), arithmetic mean aggregate mass diameter (MAMD), and the coarse‐to‐fine index (CFI = % WSA > 1.00 mm / % WSA < 0.063 mm). Analysis of WSA data indicated that the relative soil erodibility ranking, from high to low, would be: Lualualei Vertisol > Molokai Oxisol > Kaneloa Oxisol. Discriminant analysis using GMAD and % WSA > 1.00 mm correctly classified 55 of 56 soil samples into their respective soil series.     

Validation of a tomato-specific gene, LAT52, used as an endogenous reference gene in qualitative and real-time quantitative PCR detection of transgenic tomatoes

Toward the development of reliable qualitative and quantitative Polymerase Chain Reaction (PCR) detection methods of transgenic tomatoes, one tomato (Lycopersicon esculentum) species specific gene, LAT52, was selected and validated as suitable for using as an endogenous reference gene in transgenic tomato PCR detection. Both qualitative and quantitative PCR methods were assayed with 16 different tomato varieties, and identical amplified products or fluorescent signals were obtained with all of them. No amplified products and fluorescent signals were observed when DNA samples from 20 different plants such as soybean, maize, rapeseed, rice, and Arabidopsis thaliana were used as templates. These results demonstrated that the amplified LAT52 DNA sequence was specific for tomato. Furthermore, results of Southern blot showed that the LAT52 gene was a single-copy gene in the different tested tomato cultivars. In qualitative and quantitative PCR analysis, the detection sensitivities were 0.05 and 0.005 ng of tomato genomic DNA, respectively. In addition, two real-time assays employing this gene as an endogenous reference gene were established, one for the quantification of processed food samples derived from nontransgenic tomatoes that contained degraded target DNA and the other for the quantification of the junction region of CaMV35s promoter and the anti-sense ethylene-forming enzyme (EFE) gene in transgenic tomato Huafan No. 1 samples. All of these results indicated that the LAT52 gene could be successfully used as a tomato endogenous reference gene in practical qualitative and quantitative detection of transgenic tomatoes, even for some processed foods derived from transgenic and nontransgenic tomatoes.