Detection of sesame seed DNA in foods using real-time PCR

The detection of potentially allergenic foods, such as sesame seeds, in food products is a major concern for the food-processing industry. A real-time PCR method was designed to determine if sesame seed DNA is present in food products. The PCR reaction amplifies a 66-bp fragment of the sesame seed 2S albumin gene, which is detected with a sesame-specific, dual-labeled TaqMan probe. This reaction will not amplify DNA derived from other seeds present in baked goods, such as pumpkin, poppy, and sunflower seeds. Additionally, this assay will not cross-react with DNA from several tree nut species, such as almond, Brazil nut, cashew, hazelnut, and walnut, as well as four varieties of peanut. This assay is sensitive enough to detect 5 pg of purified sesame seed DNA, as well as sesame seed DNA in a spiked wheat cracker sample.     

Validation and comparison of a sandwich ELISA,two competitive ELISAs and a real-time PCR method for the detection of lupine in food

Methods applied in food allergen analysis should be specific, sensitive and applicable to both raw and highly processed foods. The performance of the most commonly used methods, ELISA and real-time PCR, may, however, be influenced by food processing steps, e.g., heat treatment. The present study compares the applicability of four in-house developed methods, one sandwich ELISA, two competitive ELISAs and a real-time PCR method, for the detection of lupine in four different food matrices, comprising bread, biscuits, rice patties and noodles. In order to investigate the influence of food processing on the detectability, not only the heat treated model foods but also the corresponding doughs were analysed. The sandwich ELISA proved to be the most sensitive method. The LOD was found to be 10 ppm lupine, independent from the food matrix and independent if the dough or the heat treated food was analysed. In addition, the methods were applied to the analysis of commercial foodstuffs differing in their labelling.     

Food matrix standards for the quantification of allergenic food ingredients using real-time PCR

For the quantification of food allergens by real-time polymerase chain reaction (PCR), food matrix standards with defined levels of spiked allergenic food ingredients can be used. The production and homogeneity testing of selected materials as sausages, cookies and sauce hollandaise powder is described. Except for egg and milk, all relevant allergenic ingredients were spiked to each material. Allergens were spiked and quantified as food ingredients, for example, peanut or lupine flour, at levels of 5–400 mg/kg. Material with sufficient homogeneity could be produced even at low levels of 5–10 mg of the allergenic ingredient per kilogram. The effect of the food matrix on allergen quantification was checked. The bias caused by this effect was in an acceptable range for the tested materials. The materials produced within this study were used as samples and for calibration in inter-laboratory validation studies for the quantification of allergenic food ingredients by real-time PCR. The results of this study are a contribution how to produce such reference materials for allergen analysis in the near future. Before threshold or action values of allergens in food are set, the availability of reference materials is essential.     

芥末等8种食物过敏原的多重PCR检测技术

依据芥末Sin a I基因、羽扇豆核糖体内转录间隔区(ITS)基因、胡桃Jug r 2基因、榛果Oleosin基因、芹菜Mtd基因、杏仁Pru du 1基因、燕麦Avenin基因和芝麻2S albumin mRNA基因设计的特异性引物序列,在普通PCR方法的基础上,通过2组4重PCR扩增,建立了同时检测芹菜、杏仁、燕麦、芝麻、芥末、羽扇豆、胡桃和榛果等8种食物过敏原的方法。该方法特异性强,灵敏性高,可应用于食品中多种食物过敏原的快速检测和监控。     

Detection of celery ( Apium graveolens ), mustard ( Sinapis alba , Brassica juncea , Brassica nigra ) and sesame ( Sesamum indicum ) in food by real-time PCR

Legislation requires labelling of foods containing allergic ingredients, amongst them celery, mustard and sesame. Here we present robust quantitative and sensitive methods for real-time PCR detection of celery, mustard (Sinapis alba and Brassica sp.) and sesame in food. The development of the DNA-based assays was part of an effort to generate alternative detection methods for allergens for which effective protein-based assays are lacking. The celery and sesame methods were specific for the celery mannitol dehydrogenase gene and the sesame allergen encoding 2S albumin gene, respectively, when tested against a range of plant materials. The mustard method was specific for the allergen encoding sinA gene and its homologues present in different Brassica sp. All primer probe pairs gave high amplification efficiency and sensitivities below approximately ten molecules of purified template DNA. These DNA-based detection methods will constitute supplementary and complementary methods to the traditional protein-based methods. Laboratories may choose different analysis formats depending on the food matrix, the availability of specific tests and the performance characteristics of the tests.     

Quantification of lupine (Lupinus angustifolius) in wheat flour using real-time PCR and an internal standard material

In the European Union, labelling of the 14 food allergens listed in Annex IIIa of Directive 2000/13/EC is mandatory. The implementation of upper limits for these allergens is under discussion. Therefore, quantitative analytical methods will be needed to verify compliance with regulatory requirements and to provide an improved basis for the legal assessment of allergen labelling. In this study, the lupine flour content in wheat flours was determined using real-time PCR and statice seeds as internal standard material. The method proved to be applicable to the quantification of lupine contents from 1 to 10?mg/kg, which is in the range relevant for allergic consumers.     

Duplex real-time PCR method for the detection of sesame (Sesamum indicum) and flaxseed (Linum usitatissimum) DNA in processed food products

The development of a duplex real-time polymerase chain reaction (PCR) method allowing the simultaneous detection of sesame and flaxseed DNA in commercial food products is described. This duplex real-time PCR technique is based in the design of sesame- and flaxseed-specific primers based on the ITS1 region and two TaqMan fluorescent probes. The method was positive for sesame and flaxseed, and showed no cross-reactivity for all other heterologous plant and animal species tested. Sesame and flaxseed could be detected in a series of model samples with defined raw and heat-treated sesame in flaxseed, and flaxseed in sesame, respectively, with detection limits of 1.3 mg kg^?1 for sesame and 1.4 mg kg^?1 for flaxseed. The applicability of the assay for determining sesame and flaxseed in different food matrices was investigated by analysing a total of 238 commercial foodstuffs. This PCR method is useful for highly selective and sensitive detection of traces of sesame and flaxseed in commercial food products.     

食品中杏仁过敏原成分的检测

目的：建立检测食品中桃仁、杏仁过敏原成分的荧光PCR 方法,比较国外3 种ELISA 试剂盒效果。方法：针对杏仁Pru du1 基因设计引物及探针,建立荧光PCR 方法。利用杏仁过敏原参考物质对3 个品牌的ELISA试剂盒的回收率进行比较。结果：建立的荧光PCR 方法,具有很好的特异性;灵敏度为10mg/kg。结论：桃仁及杏仁过敏原成分荧光PCR 检测方法特异性好、灵敏度高,对食品中过敏原的检测有重要的实际意义。     

Optical thin-film biochips for multiplex detection of eight allergens in food

An innovative method for the rapid detection of food allergens is developed and validated. Here we reported the development of silicon-based optical thin-film biochip technology that simultaneously permits visible detection of eight food allergens including celery, almond, oat, sesame, mustard, lupine, walnut and hazelnut on the basis of two tetraplex PCR systems. The biochip detection time was about 30 min after PCR amplification. Briefly, the optical thin-film biochip detects the presence of PCR fragment targets by enzymatically converting the formation of nucleic acid hybrids to molecular thin films. The mass contributed by the thin film alters the interference pattern of light on the biochip surface, resulting in a visible color change on the chip surface. Therefore, this assay permits sensitive, specific and high-throughput detection of allergens in food samples.     

Two tetraplex real-time PCR for the detection and quantification of DNA from eight allergens in food

According to the EU and Swiss legislation, food has to be labelled for allergens to enable allergic consumers to avoid such food and its products. To provide efficient and reliable methods, two novel quantitative multiplex real-time polymerase chain reaction systems were developed and validated. They simultaneously determine DNA of peanut, hazelnut, celery, soy, egg, milk, almond and sesame, respectively. The tests exhibit good specificity and sensitivity in the range of 0.01%. Due to low DNA amounts, lower sensitivities for egg and milk were obtained. First comparisons of ELISA results with PCR results suggest a qualitative accordance, but a low correlation of quantitative results.     

First ring-trial validation of real-time PCR methods for the quantification of allergenic food ingredients

The first interlaboratory validation of two food allergen quantification methods using real-time PCR is described. Methods for the specific detection and quantification of soybean and white mustard in boiled sausages were used. Matrix-based calibrants spiked with defined amounts of soybean and white mustard were applied for quantitative evaluation. The lowest spike level of 10?mg soybean and white mustard per kilogram could reproducibly be detected. Recovery in spiked sausages was between 82 and 99?% for soy and between 80 and 93?% for mustard. Reproducibility standard deviation was in the range that would be acceptable, for example, for quantitative GMO analytical methods (<35?%).     

Application of real-time PCR for tree nut allergen detection in processed foods

Abstract

Currently, food allergies are an important health concern worldwide. The presence of undeclared allergenic ingredients or the presence of traces of allergens due to accidental contamination during food processing poses a great health risk to sensitized individuals. Therefore, reliable analytical methods are required to detect and identify allergenic ingredients in food products. Real-time PCR allowed a specific and accurate amplification of allergen sequences. Some processing methods could induce the fragmentation and/or degradation of genomic DNA and some studies have been performed to analyze the effect of processing on the detection of different targets, as thermal treatment, with and without applying pressure. In this review, we give an updated overview of the applications of real-time PCR for the detection of allergens of tree nut in processed food products. The different variables that contribute to the performance of PCR methodology for allergen detection are also review and discussed.     

基于分子生物学技术检测食物过敏原的研究进展

食物过敏现象越来越普遍,已经严重影响了部分人群的生活质量甚至危及生命,国际上有关食物过敏事件的报道屡见不鲜。如何快速、准确、高通量、低成本地检测食物中的过敏原成分,已成为监管部门和食品企业的关注焦点。本文综述了以酶联免疫吸附法、PCR法、实时荧光定量PCR法、环介导等温扩增技术及生物芯片技术为主的几种分子生物学检测技术检测食物过敏原的方法,并对这几种方法的准确性、重复性等进行比较,探讨了食品中过敏原检测方法的发展前景。与传统过敏原检测方法相比,现行生物芯片技术具有高通量、对单一样品同时检测多种过敏原成分的特点,将成为一种新颖有效的过敏原检测工具。     

A survey of the bacteriological quality of preroasted peanut, almond, cashew, hazelnut, and brazil nut kernels received into three Australian nut-processing facilities over a period of 3 years

There is little information about bacteriological quality of preroasted kernels available in the public domain. An investigation of the bacteriological quality of preroasted peanut, almond, cashew, hazelnut, and Brazil nut kernels received into three Australian nut-processing facilities was performed over a period of 3 years. A total of 836 samples were analyzed for aerobic plate count, and 921 samples for Salmonella and Escherichia coli. The 921 samples included 653 peanut, 100 cashew, 60 almond, 60 Brazil nut, and 48 hazelnut kernels. There was no E. coli detected in any sample. Salmonella subsp. II (Fremantle) was detected in one raw almond sample. The aerobic plate count percentages of positive samples with counts above the detection level of the plating method used (100 CFU/g) for peanuts, almonds, cashews, hazelnuts, and Brazil nuts were 84, 78, 74, 50, and 45%, respectively. Of the samples containing more than this detection limit, the means were 4.5, 4.4, 3.1, 2.5, and 3.8 log CFU/g respectively. Although roasted kernel quality was not within the scope of this survey, raw microbial bioload would be expected to reduce on roasting. The bacteriological quality of preroasted peanut, almond, cashew, hazelnut, and Brazil nut kernels received into nut-processing facilities in Australia does not appear to suggest a public health concern.     

Selection of a Taxon-Specific Reference Gene for Qualitative and Quantitative PCR Detection of <Emphasis Type="Italic">Carthamus tinctorius</Emphasis>

Safflower (Carthamus tinctorius) is an emerging model plant for the transgenic modification of fatty acid composition and the production of pharmaceuticals, proteins, or enzymes. Safflower is also a traditional Chinese medicine and is often used as a fake saffron product. Reliable detection of an endogenous reference gene is indispensable for the supervision of genetically modified safflower. Such an endogenous reference gene can also be used to specifically identify safflower ingredient in complex mixtures such as medicine or food. In this study, we identified and validated the CTOS gene as an endogenous reference for safflower. Conventional and real-time polymerase chain reaction (PCR) methods for detecting the CTOS gene sequence showed high interspecies specificity and intra-species stability. The lowest copy number detectable by conventional PCR was 10 haploid copies. The limit of detection and limit of quantification for the real-time PCR assay were estimated to be five and 40 haploid genome copies, respectively. Standard curves established for the real-time PCR assay exhibited good linearity (R ² > 0.99) between the cycle threshold (Ct) values and the initial template copies. The developed conventional and real-time PCR assays were validated in routine analysis of the safflower ingredient in commercial Chinese medicines. In conclusion, the developed quantitative PCR methods were sufficiently specific and sensitive to be used in safflower genomic DNA quantification and safflower ingredient identification.     

A cotton‐specific gene,stearoyl‐ACP desaturase,used as a reference for qualitative and real‐time quantitative polymerase chain reaction detection of genetically modified organisms

Biotechnology has permitted the modification of agricultural materials in a very precise way to improve productivity and yields. Polymerase chain reaction (PCR)‐based methods have been the first choice of most analytical laboratories for routine use in the detection of genetically modified organisms (GMO) and their derived products. These methods rely on the amplification of transgenic sequences and quantification of the transgenic DNA by comparison with an amplified reference gene. This paper describes the specific primers and probe for the cotton stearoyl‐ACP desaturase (sad1) gene, and PCR cycling conditions suitable for the use of this sequence, which acts as an endogenous reference gene in both qualitative and quantitative PCR assays. The two methods were tested with 18 cotton varieties and identical amplification products were obtained with all of them. No amplification products were detected when DNA samples from other species, including soybean, rapeseed, tobacco, maize, tomato, potato, cucumber, pea, red pepper, sunflower, sesame, rice, peach, banana, apple, pumpkin, barley and carrot, were used as templates, which demonstrates that this system is specific for cotton. In real‐time quantitative PCR analysis, the detection limit was as low as 6 pg of DNA, which indicates that this method is suitable for application to processed food samples that contain very low copies of target DNA. Southern blot analysis confirmed that the sad1 gene was a single copy in the tested cotton varieties. Copyright © 2006 Society of Chemical Industry     

芝麻过敏原PCR检测方法

芝麻是重要的食品过敏原之一,微小剂量即可引起严重的过敏反应。针对芝麻过敏蛋白-2S白蛋白的基因序列设计PCR扩增引物,建立并优化芝麻过敏原检测的普通聚合酶链式反应(PCR)和实时荧光定量聚合酶链式反应(Real-time PCR)方法。结果表明,两种方法的最低检测限分别为0.1,0.01ng DNA,该方法特异性良好,成本较低,具有一定的应用价值。     

实时荧光P C R 法检测食品中芝麻过敏原成分

本实验探讨采用实时荧光定量PCR 方法检测食品中的芝麻成分,结果表明：采用芝麻的引物和探针进行扩增时;22 种样品经实时PCR 扩增,只有白芝麻和黑芝麻产生荧光信号,其余样品均不产生荧光信号。灵敏度实验结果表明能检测到10-5 稀释度的4.4 ×10-12g DNA的量,定量关系式为：y=－ 3.472018x+18.851009,R2=0.993088。这两对引物和探针具有极高的灵敏度和扩增效率,符合痕量检测要求。     

An event-specific real-time PCR detection system for the transgenic rice line 114-7-2 of producing functional human serum albumin

Transgenic rice 114-7-2 is a newly developed transgenic rice line of producing human serum albumin (HSA). It has attracted much attention because of its economic potential. This paper was designated to discover the integration site of the transgenic HSA rice line 114-7-2 and to establish event-specific methods for qualitative and quantitative detection of the transgenic HSA rice based on the border junction fragment. One gene fragment of 5′ flanking region was successfully isolated using the TAIL-PCR methods. The fragment sequence showed that a 454-bp junction fragment contained 75 bp of T-DNA sequence and 379 bp of rice genome DNA, which is located in chromosome 4. Event-specific real-time PCR method for HSA rice line 114-7-2 was established with the primers (HSA-F/HSA-R) and the probe (HSA-P) targeting the 454-bp junction region. The qualitative PCR assay showed the limit of detection was 0.01 %. In the event-specific quantitative detection method, the LOQ for 114-7-2 HSA rice was estimated to be 0.025 ng or 50 copies. The method developed in this study is highly specific, sensitive, and reliable for transgenic HSA rice sample detection.     

Development and Validation of Two Competitive ELISAs for the Detection of Potentially Allergenic Lupine (Lupinus Species) in Food

The paper presents two competitive enzyme linked immunosorbent assays (ELISAs) for the detection of lupine proteins in foods. The ELISAs are based on anti-lupine antibodies produced in a rabbit (IgG) and in a chicken (IgY), respectively. Cross-reactivity tests with 33 foods of plant origin revealed that both ELISAs show slight cross-reactivity (≤1.5 %) with pecan nut. They do, however, not cross-react with 32 species potentially used in lupine containing foodstuffs. Both ELISAs detect proteins from white (Lupinus albus) and blue (Lupinus angustifolius) lupine and, with a lower sensitivity, proteins from yellow (Lupinus luteus) lupine. Matrix effects could be eliminated by diluting the sample extracts 1:10 before loading them onto the microtiter plate. The IgG ELISA allows the detection of 50 ppm white lupine flour in bread, vegetarian patties and rusk. With the IgY ELISA, lupine can be detected in vegetarian patties and bread at the 50 ppm spiking level and in rusk at the 100 ppm spike level.