Simultaneous detection of DNA from 10 food allergens by ligation-dependent probe amplification

The simultaneous detection of DNA from different allergenic food ingredients by a ligation-dependent probe amplification (LPA) system is described. The approach allows detection of several targets in a one-tube assay. Synthetic oligonucleotides were designed to detect DNA from peanuts, cashews, pecans, pistachios, hazelnuts, sesame seeds, macadamia nuts, almonds, walnuts and brazil nuts. The specificity of the system was tested with DNA from more than 50 plant and animal species. The sensitivity of the method was suitable to detect allergenic ingredients in the low mg kg^?1 range. The limit of detection (LOD) for single allergens in different food matrices was 5 mg kg^?1. The novel analytical strategy represents a useful tool for the surveillance of established legislation on food allergens within the European Union.     

A novel real-time polymerase chain reaction method for the detection of pecan nuts in food

A real-time PCR-based method for the detection of the pecan (Carya illinoiensis) component in food is described. The method consists of DNA isolation by chaotropic solid phase extraction and the subsequent PCR with pecan-specific primers and a TaqMan fluorescent probe. The primers and the probe are targeted to the putative gene for allergenic vicilin-like seed storage protein of pecan. The method was positive for 10 pecan varieties and negative for all other tested plant materials used in food industry, including walnut. The intrinsic detection limit of the method was 1 pg pecan DNA which corresponds to 1.2 haploid genome copies. Using a series of model pastry samples with defined pecan contents, a practical detection limit of 0.01% (w/w) pecan was estimated. Practical applicability of the PCR method was tested by the analysis of 13 food samples; no discrepancies between the declared and detected pecan contents were found. The presented PCR method is useful for sensitive and selective detection of pecans in food samples and can be performed in one working day.     

Detection of allergenic parvalbumin of Atlantic and Pacific herrings in fish products by PCR

The conventional polymerase chain reaction (PCR) method to detect the major allergenic protein parvalbumin beta 2 of Atlantic herring (Clupea harengus) and Pacific herring (Clupea pallasii) was developed. The specific set of primers for the amplification of the partial genomic sequence of the pvalb 2 gene encoding the main fish allergen of both herrings was designed and applied to the investigation of 24 commercial fish products. The targeted amplicon size was 189 bp of pvalb 2 gene of Atlantic herring and Pacific herring. As the internal amplification control, the DNA of 18S rRNA gene for eukaryotes (141 bp) was successfully used. The specificity of designed primer pair using 26 various fish species was assessed. The intrinsic detection limit was 10 pg µl^?1 of the present specific DNA. Atlantic herring or Pacific herring allergenic parvalbumins were detected in 22 investigated fish products in conformity with the package declaration. Two fish products were negative in spite of the declaration. The proposed PCR method is specific enough and can be used for the detection of Atlantic and Pacific herrings’ major allergen parvalbumin beta 2 in fish food products.     

A novel real-time polymerase chain reaction method for the detection of macadamia nuts in food

A real-time PCR-based method for the detection of macadamia nuts (fruits of Macadamia integrifolia or M. tetraphylla or their hybrids) in food products is described. The method consists of DNA isolation by chaotropic solid phase extraction and subsequent PCR with macadamia-specific primers and a TaqMan fluorescent probe. The primers and the probe were targeted to the gene encoding for vicilin precursor. The method was positive for M. integrifolia and M. tetraphylla and negative for 16 other plant species used in food industry, including peanuts, walnuts, hazelnuts, almonds, pistachio nuts, cashew nuts, Brazil nuts, and chestnuts. The DNA-based detection limit of the method was 1.45 pg. Using a series of model samples with defined macadamia nut contents, a practical detection limit of 0.02% (w/w) macadamia nuts was determined. Practical applicability of the PCR method was tested by the analysis of 14 confectionery samples. For all of the samples, results conforming to the labeling were obtained. The presented PCR method is useful for relatively fast, highly selective, and moderately sensitive detection of macadamia nuts in food samples.     

A novel real-time polymerase chain reaction (PCR) method for the detection of walnuts in food

A real-time polymerase chain reaction (PCR)-based method for the detection of the walnut (Juglans regia) component in food is described here. The method consists of DNA isolation by chaotropic solid phase extraction and the subsequent PCR with walnut-specific primers and a TaqMan fluorescent probe. The primers and the probe are targeted to the jug r2, a major allergen gene of walnut. The method was positive for 8 varieties of walnut and negative for all other tested plant materials used in food industry, including pecan nuts. The intrinsic detection limit of the method was 0.24 ng walnut DNA. Using a series of model pastry samples with defined walnut contents, a practical detection limit of 0.01% walnut content was estimated. Practical applicability of the PCR method was tested by the analysis of 13 food samples (bakery and confectionery products), out of which two cakes were found to contain walnuts although they were not adequately labelled. The presented PCR method is useful for sensitive and selective detection of walnuts in food samples and can be performed in one working day.     

Development of a Competitive ELISA for the Detection of Pecan (Carya illinoinensis (Wangenh.) K. Koch) Traces in Food

Pecan nuts are included among the so-called big-eight food allergens, which are responsible for 90% of food allergies. However, unlike other tree nuts such as Brazil nut and hazelnut, studies on pecan from the point of view of allergies are scarce, and only a few analytical methods to detect its presence in food have been published. The objective of the present study was to develop an enzyme-linked immunosorbent assay (ELISA) method to detect traces of pecan in foods. Therefore, pecan-specific polyclonal antibodies were developed in rabbits and characterized for their specificity to pecan proteins and for their selectivity in challenging studies with other food ingredients and allergens. In addition, the research was complemented with more specific analyses like sodium dodecyl sulfate polyacrylamide gel electrophoresis to characterize the protein standard and western blot to identify the reactive proteins. By applying the selected conditions, the data were fitted to a four parameter logistic curve, which gave a R square of 0.999, and IC50 and IC80 values of 5.0 and 2.2 ng/mL, respectively. This highly sensitive method allows the detection of trace amounts of pecan protein within complex food matrices, which can provide the food industry with a useful tool to comply with the requirements enforced by regulatory agencies.     

Simultaneous detection of allergenic fish,cephalopods and shellfish in food by multiplex ligation-dependent probe amplification

People suffering from food allergy rely on correct food labelling as the ingestion of minimal amounts of the respective allergen can trigger severe allergenic reactions. Probes for the detection of DNA from allergenic fish, shellfish and cephalopod species in food using multiplex ligation-dependent probe amplification were developed. The specificity and the sensitivity of the detection system were investigated. The limit of detection was 20 mg kg^?1 for scallop, fish and bivalve species and 100 mg kg^?1 for cephalopod, gastropod and crustacean species using self-prepared sushi spiked with the analytes in different concentration levels. The analysis of 10 commercial food samples demonstrates the applicability of the developed method and its suitability for food quality control. Therefore, the method can be used to monitor the compliance with labelling rules regarding food allergens.     

Development and validation of a SYBR‐Green I Real‐Time PCR test to detect bivalves including Mytilus species in foods

The incidence of allergy to seafood, and in particular to molluscs is second only to that of nuts. To protect consumers, the regulators of food products insisted on identifying molluscs as allergens. The aim was to develop quantitative assay for the presence Mytilus species in processed food products. The chosen platform was real‐time PCR (qPCR) targeting either the gene encoding mitochondrial cytochrome C oxidase I or the nuclear gene encoding β‐actin. Recombinant plasmids containing each of target regions were used as a reference for quantification purposes. Limit of detection (LOD) and of quantification (LOQ) were determined. Spiked food samples containing 50–500 μg g^?1 of Mytilus chilensis were analysed both by qPCR and by ELISA. The former assay gave a positive outcome over this range, whereas the latter was sensitive down to a concentration of 125 μg g^?1.     

A novel real-time polymerase chain reaction (PCR) method for the detection of hazelnuts in food

A real-time PCR (polymerase chain reaction)-based method for the detection of hazelnuts (nuts of Corylus avellana or C. maxima) in confectionery and bakery products is described. The method consists of DNA isolation by chaotropic solid phase extraction and the subsequent PCR with hazelnut-specific primers and a TaqMan fluorescent probe. The primers and the probe are targeted to the hsp1 gene encoding for a low molecular weight heat-shock protein. The method was positive for five hazelnut varieties approved in Slovakia and negative for all other tested plant materials used in food industry including peanuts, walnuts, almonds, pistachio nuts, cashews and chestnuts. The intrinsic detection limit of the method was 13 pg hazelnut DNA, which corresponds to approximately 27 genome equivalents (1C). Using a series of model pastry samples with defined hazelnut contents, a practical detection limit of 0.01% (w/w) hazelnut was determined. Practical applicability of the PCR method was tested by the analysis of 20 food samples (confectionery and bakery products) along with ELISA. For all of the food samples, identical results were obtained by both methods, which conformed to the labelling. The presented PCR method is useful for sensitive and selective detection of hazelnuts in food samples and can be performed in one working day.     

Effect of relative humidity,storage days,and packaging on pecan kernel texture: Analyses and modeling

The studies expounding on the effects of storage conditions on texture changes are limited. The researchers have been proposing methods to measure pecan texture instrumentally. But current protocols and/or attributes fail to address huge variability during experimentation. Additionally, there are no predictive models to estimate changes in pecan texture during storage. This study addresses all the above concerns and investigates the effects of different relative humidity (RH, 30–90%) and packaging material (Polyethylene-Nylon [PEN], polypropylene [PP], low density polyethylene [LDPE], and metallic laminates [ML]) on pecan texture, introducing a rift ratio (F/H or fracturability to hardness ratio) to address variability in the data and predictive model to estimate changes in the textural attribute of pecans during storage. The textural analysis was conducted on pecan cores and intact pecans to measure the area under curve, fracturability, hardness, cohesiveness, chewiness, springiness, and rift ratio. It was observed that values for the rift ratio obtained using the intact pecan method had high R² (0.72) as compared to the rest of the textural attributes. A three-parameter logistic model was employed to predict pecan texture during storage. The pecans stored at 75, 80, and 90% reached the rift ratio (F/H) of 0.5 at approx. 115, 3, and 0.15 days (~ 4 hr), respectively. Similarly, pecans stored in LDPE, PP, and PEN packs at 80% reached rift ratio (F/H) of 0.5 at approx. 26, 57, and 78 days, respectively. The presence of any kind of package delayed fracturability loss by at least eight folds at 80% RH. The pecans stored in ML did not experience a significant change in textural attributes     

A novel real-time polymerase chain reaction method for the qualitative detection of pistachio in food

In order to provide an appropriate method for the detection of pistachio (Pistacia vera) in food products, a novel real-time PCR was developed. The pistachio-specific primers and the TaqMan fluorogenic probe were designed to target the internal transcribed spacer between 18S ribosomal RNA and 5.8S ribosomal RNA genes. Using dilutions of the pistachio DNA, the intrinsic detection limit of the method was determined to be 0.012 pg. At specificity testing, the method was positive for 11 pistachio varieties and negative for 26 plant and animal species used in food industry. A detection limit of 0.0004% (w/w) was determined for pistachio nuts in model pastry. Practical applicability of the elaborated method was tested by the analysis of 44 food samples, out of which 7 food products were identified as containing undeclared pistachio. The developed real-time PCR may be utilized for sensitive and selective detection of pistachio in food products.     

双重实时荧光PCR法测定食品中大豆、小麦过敏原

目的:为食品监管部门有效检测食品中大豆、小麦过敏原提供技术支撑。方法:分别依据小麦醇溶蛋白基因及大豆Lectin基因为模板设计并创建TaqMan探针双重荧光PCR方法,大豆Lectin基因检测采用FAM标记,小麦醇溶蛋白基因检测采用HEX标记,同时以真核生物18S rRNA作为内参基因确保检测体系的有效性。结果:所创建的实时多重TaqMan探针PCR体系对大豆、小麦过敏原之外的物种成分无荧光扩增;大豆、小麦混合样品的检出限均能达到0.01%(质量分数)。结论:所创建的实时多重TaqMan探针PCR体系针对大豆、小麦过敏原有高特异性,可用于食品中过敏原大豆、小麦成分的同步快速检测。     

Effect of Processing on the Detectability of Pecan Proteins Assessed by Immunological and Proteomic Tools

The present study evaluates the effect of food processing on the antigenicity of pecan proteins as measured by enzyme-linked immunosorbent assay (ELISA). In addition, proteomic tools were used to identify potential pecan markers suitable for confirming the presence of pecan proteins in food and validating new methods developed to detect traces of the commodity. To assess the effects of processing on protein stability and antigenicity, pecan nuts were submitted to heat treatments and extracts were analysed by ELISA, sodium dodecyl sulphate polyacrylamide gel electrophoresis and Western blot. The ELISA method was able to detect pecan traces even after submitting the commodity to rigorous treatments, though these treatments affected the detectability to varying degrees. Proteomic assessment showed that the majority of pecan proteins were matched by homology to walnut proteins, which are more abundantly populated in the protein sequence databases. However, there were a few important exceptions: 7S vicilin, 11S legumin and putative allergen I1, unambiguously identified as pecan in origin. Interestingly, putative allergen I1 offered unique analytical advantages to be used as a pecan marker for validation and confirmation purposes.     

FACTORS INFLUENCING FUNGAL QUALITY OF PECANS STORED AT REFRIGERATION TEMPERATURES

TWO factors greatly affecting the growth of fungi on stored pecan nuts (Curya illioensis) are moisture content and temperature. Experiments designed to simulate conditions of commercial practice were carried out to determine the effects of sap (naturally occurring) moisture and superficial or imbibed moisture, i.e., moisture originating from sources other than the tree, on survival of fungi on stored pecans. A second objective was to determine the effect of temperature of storage on the rate of equilibration of moisture in pecans. Of particular interest was the effect of this rate of change on populations of naturally occurring fungal contaminants. In-shell pecans were stored in Kraft paper and plastic bags at ?18, ?6.5, and 0°C for periods of 6, 12, and 24 wk. Results indicate that pecan kernels with low sap and low superficial moisture at the time of harvest are less likely to support fungal growth than are kernels with high sap moisture, or low sap moisture but high superficial moisture. Fungi are capable of growing on pecans at 0°C and to a less extent at ?6.5°C after prolonged storage. Rapid reduction of moisture content accompanied by ventilation in chambers with air having relative humidities in the range 60–68% is most desirable for preserving pecans against fungal deterioration.     

Polymerase chain reaction (PCR) for the detection of celery (<Emphasis Type="Italic">Apium graveolens</Emphasis>) in food

A method based upon polymerase chain reaction (PCR) for the detection of celery (Apium graveolens) in food was developed. The method involves DNA isolation by chaotropic or non-chaotropic solid-phase extraction and PCR with primers oriented to the sequence of the nuclear gene encoding mannitol dehydrogenase. The PCR method was shown to be specific for celery, producing a 279 bp fragment with four celery varieties and negative results with other species commonly present together with celery in food products (16 samples). The detection limit of PCR was 490–1530 pg DNA, which corresponds to 10² genome copies. When evaluated with model samples of celery in meat pâtés, a detection limit of 0.1% (w/w) was determined. When used to analyse food products from the market (dried vegetable seasonings, dehydrated bouillons), all four products declared to contain celery were correctly identified as positive and all three products in which celery was not declared were identified as negative.     

Monitoring of Bt11 and Bt176 genetically modified maize in food sold commercially in Brazil from 2005 to 2007

BACKGROUND: The first genetically modified (GM) maize lines were approved for trading in Brazil after December 2007 and they were T25, MON810, Bt11, NK603 and GA21. The polymerase chain reaction (PCR) method was employed to monitor the presence of Bt11 and nested PCR was used to detect the presence of Bt176 in 81 maize‐derived products (maize flour, corn meal, maize flour flakes and polenta) that were sold in Brazilian market from 2005 to 2007, before the release of GM maize in Brazil. RESULTS: The PCR detection limit for Bt11 was 10 g kg^?1 and for nested PCR of Bt176 it was 1 g kg^?1. All Brazilian samples analyzed showed no positive signal for these GM maize events. CONCLUSION: Bt11 and Bt176 GM maize lines were not detected by specific PCR in 81 maize‐derived food samples sold in Brazil from 2005 to 2007, before the commercial release of GM maize in Brazil. These Brazilian food industries were in compliance with the rules stipulated by the current legislation with respect to consumer requirements about GMO labeling. Copyright © 2010 Society of Chemical Industry     

Signal amplification using colloidal gold in a biolayer interferometry-based immunosensor for the mycotoxin deoxynivalenol

Deoxynivalenol (DON) is a toxin produced by certain species of Fusarium fungi that can infest wheat, barley and corn. The fungi cause diseases in crops worldwide and some of the secondary metabolites, such as DON, can adversely affect animal health and food safety. To monitor DON in wheat rapidly, a biosensor using the principle of biolayer interferometry (BLI) was developed. The signal from the sensor was substantially amplified through the use of a primary antibody–colloidal gold conjugate. The amplification was much greater in the presence of wheat matrix than in buffered solution, suggesting matrix components may have contributed to the enhancement. The improved signal provided by the amplification allowed for the development of rapid qualitative and quantitative assays. The limit of detection of the method was 0.09?mg?kg^?1; the limit of quantitation was 0.35?mg?kg^?1. Recovery from wheat spiked over the range from 0.2 to 5?mg?kg^?1 averaged 103% (RSD?=?12%). The quantitative assay compared favourably (r ^2?=?0.9698) with a reference chromatographic method for 40 naturally contaminated wheats. The qualitative assay was able to classify accurately the same group of 40 samples as either above or below a 0.5?mg?kg^?1 threshold. These results suggest that the BLI technique can be used to measure DON in wheat rapidly.     

Microbial, Compositional, and Other Quality Characteristics of Pecan Kernels Stored at −20°C for Twenty-five Years

Sensory, microbial, and compositional characteristics of pecans hermetically sealed in cans and stored at ?20°C for 25 yr were compared to those of pecans harvested in 1986 and stored at ?20°C for 10 mo. Total aerobic and yeast/mold populations were 10²?10³ CFU/g in stored and 1986 crop nuts. Peroxides were not detected, and no difference in iodine values and free fatty acid contents were observed between 25-yr-old and 1986 crop nuts. Although subjective analyses revealed that texture changed during storage, other sensory quality characteristics (color, flavor, appearance) were not different from pecans harvested in 1986. It is concluded that low temperature (?20°C) storage will maintain quality of pecans packaged in hermetically sealed under ambient air in containers for up to 25 yr.     

Pecan nuts: A review of reported bioactivities and health effects

BackgroundFood choices represent a highly significant approach to combat human obesity. Dietary intake of lipids, especially polyunsaturated and monounsaturated fatty acids, is gaining popularity in the effort to reduce or eliminate the occurrence of obesity. Pecan (Carya illinoinensis) nuts are an abundant source of these dietary fatty acids. Moreover, they are a rich source of epigallocatechin-3-gallate (EGCG), a polyphenol with a variety of health-beneficial properties.Scope and approachIn this review, we summarize the literature reports examining physiological effects associated with pecan nuts consumption and described effects of their bioactive constituents.Key findings and conclusionsThe growing body of evidence suggests including pecan nuts into obesity management strategies. The consumption of pecan nuts can mitigate inflammation by reducing the extent of the synthesis of inflammatory mediator molecules. Pecan nuts can also counteract the pro-inflammatory effects of a diet rich in commonly overconsumed saturated fatty acids, characteristic of the Western diet. Additionally, consumption of pecans and other nuts has been linked to reduced risk of physiological parameters associated with cardiovascular disease or metabolic disorders. Diets enriched with tree nuts and peanuts can modulate the blood level of cholesterol, adiposity, and insulin resistance. Almonds and walnuts have been so far the most studied nuts, and studies with them have led to a greater understanding of the protective effects of diverse tree nuts on human physiology. In this review, we summarize the available data indicating that pecan nuts exert similar health-promoting benefits.     

Inactivation of Salmonella on in-shell pecans during conditioning treatments preceding cracking and shelling

Studies were done to determine the effectiveness of conditioning treatments for killing Salmonella in and on immersion-inoculated and surface-inoculated in-shell pecans. Treatment of immersion-inoculated, dried, stored pecans in chlorinated water (400 μg/ml) reduced Salmonella by not more than 1.6 log CFU/g. Treatment of immersion-inoculated, dried, stored pecans in chlorinated water (200 μg/ml, 1 min) followed by soaking in water for 2 h at 21°C and treating for 10 min in water at 85 to 95°C reduced Salmonella by >5.12 log CFU/g; treatment of nuts containing a low population of Salmonella (<0.60 log CFU/g) for 15 min at 90°C failed to eliminate the pathogen. Reductions of ≥6.42 log CFU/g were achieved by treating surface-inoculated nuts in water at 90 or 95°C for 80 s; treatment of nuts containing 1.78 log CFU/g at 95°C for 10 min did not eliminate the pathogen. Salmonella on surface-inoculated in-shell pecans (kernel moisture, 4.75%; water activity, 0.62) that had been dried and stored at 4°C for 3 to 5 weeks was more resistant to conditioning treatments than was Salmonella on surface-inoculated pecans (kernel moisture, 5.60%; water activity, 0.73) that were not thoroughly dried. Conditioning treatments were less effective for killing Salmonella on immersion-inoculated pecans than on surface-inoculated pecans. Response of Salmonella to conditioning treatments varied, depending on the method of inoculation and whether nuts were dried and stored between the time of inoculation and treatment, which emphasizes the importance of following practices commonly used by commercial pecan shellers when validating the lethality of conditioning treatments.