Two quantitative hexaplex real-time PCR systems for the detection and quantification of DNA from twelve allergens in food

According to the EU and Swiss legislation, allergens in food have to be labelled. Consumers, exhibiting allergic reaction, must be able to avoid such food and its products. In order to provide efficient and reliable methods, two novel hexaplex quantitative real-time polymerase chain reaction systems were developed and validated. The first system simultaneously determines DNA contents from cashew, peanut, hazelnut, celery, soy, mustard, whereas the second system determines DNA contents from milk (beef), egg (chicken), almonds, sesame, pistachio and walnut. The two tests exhibited a good specificity and a detection limit of at least 0.1?% for all analytes. This was additionally verified using samples from proficiency tests. Application on samples from the market revealed realistic and useful information about allergen contents. Quantitative results according to weight are not possible yet.     

SYBR-Green real-time PCR approach for the detection and quantification of pig DNA in feedstuffs

A real-time polymerase chain reaction assay using primers targeting the porcine-specific mitochondrial 12S rRNA gene and universal eukaryotic primers amplifying a conserved fragment of the nuclear 18S rRNA gene has been developed for the detection and quantification of porcine DNA in food and feedstuffs. The 18S rRNA primers were used as endogenous control for the total content of PCR-amplifiable DNA in the sample. The assay was tested on DNA extracted from raw and heat-treated binary mixtures of porcine tissues in a plant matrix, and on DNA extracted from reference feedstuff samples. Analysis of experimental mixtures demonstrated the suitability of the assay for the detection and quantification of porcine DNA in mixtures containing as little as 0.1%.     

Multiplex real-time PCR for the detection and quantification of DNA from beef, pork, chicken and turkey

Many meat products are composed of two or more meat species. To determine the proportion of these meat fractions, a quantitative multiplex PCR was developed for the quantification of beef, pork, chicken and turkey. This system proved its applicability, precision and accuracy in examining different meat products from the market. Thus it allows the efficient control of composed meat products in official food control and production control laboratories.     

Multiplex real-time PCR for the detection and quantification of DNA from beef,pork, horse and sheep

Meat products are often composed of more than one meat species. A quantitative multiplex PCR was developed to determine the proportion of meat fractions of beef, pork, horse and sheep. The precision and accuracy were investigated by dilutions of DNA from all four species and examining different meat products from the market. Application of this tetraplex quantitative real-time PCR system will enable official food control and production control laboratories to efficiently investigate the composition of meat products.     

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.     

Development of a real-time PCR method for the simultaneous detection of soya and lupin mitochondrial DNA as markers for the presence of allergens in processed food

Lupin and soya are members of the Leguminosae family which are recognised as some of the richest source of vegetable proteins. Lupin- and soya-containing products are available on the EU market and could cause severe adverse reactions in allergic individuals, even if consumed at low concentrations. In this context the development of methods for reliable detection of these allergens in food products is a useful tool for the surveillance of established legislation on food labelling within the EU. This work described the development of a duplex real-time PCR method allowing the simultaneous detection of traces of lupin and soya in processed food based on a specific TaqMan® probe designed on a mitochondrial tRNA-MET gene. A set of primers and probes was designed for the amplification of a 168 and 175 bp fragment of lupin and soya mitochondrial DNA, respectively. The performance of the method was established using lupin and soya flours and cookies baked from lupin- and soya-containing dough (different concentrations and baking times). The PCR platform yielded consistent and repeatable results. The specificity of the system was tested with DNA from 28 plant species. The sensitivity of the method was suitable to detect allergenic ingredients in the low mg per kg range. Both lupin and soya at a level of 2.5 mg per kg food matrix could be detected in cookies baked at 180 °C for 10 min. The method was successfully applied to bakery (e.g. bread) and vegetarian (e.g. non-meat sausages) food products that contain or may contain soya and/or lupin as ingredient or contaminant (according to the declaration on the product label).     

Automated DNA preparation from maize tissues and food samples suitable for real-time PCR detection of native genes

There is an increasing need for high-throughput analyses of plants and food samples for the presence of specific DNA sequences, e.g. transgenic contaminations. We developed and optimized conditions for the automated isolation of DNA from several maize tissues and various edibles containing maize using the MagNA Pure LC system (Roche Applied Science). Our results show that the system provided is capable of isolating DNA from any tested source. Quantification of an endogenous gene by LightCycler real-time PCR revealed that the DNA is suitable in quality and quantity for multiple PCR analyses.     

Multiplex real-time PCR for the detection and quantification of DNA from duck, goose, chicken, turkey and pork

Meat products made from liver of poultry like duck and goose are popular and often sold as specialities for high prices. As the prices for the basic raw material are high, fraud may be attractive for producers. To prevent consumers from fraud, official control authorities survey such products. In this work, a quantitative multiplex PCR was developed determining the proportion of DNA and meat fractions of turkey, chicken, duck, goose and pork. The precision and accuracy of the PCR system was investigated. To examine the possibility of determining the meat fractions according to the recipe, reference material was produced and different liver–meat products from the market were analysed. For major components, the measurement uncertainty revealed to be at 39 %. For minor components, it was estimated to be 124 %. The results showed that this pentaplex real-time-PCR system is suitable to control the meat properties of such products although measurement uncertainty may be high.     

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。     

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.     

实时荧光定量PCR在食品检测领域中应用

实时荧光定量PCR是在定性PCR技术基础上发展起来的核酸定量技术;该技术不仅实现对DNA模板定量,且具有灵敏度高、特异性强、无污染性、及实时性和准确性等特点。该文主要介绍实时荧光定量PCR原理和在食品检测中应用,及其在食品领域发展前景。     

实时荧光定量PCR技术原理及在食品检测中的应用

实时荧光定量PCR技术是一种新兴的核酸定量分析技术,与普通PCR定性分析相比,具有简单高效、准确灵敏、实时分析等更多的优势,是一项实用性很强的技术方法。文中主要对实时荧光定量PCR技术的操作原理、荧光类型及其在食品检测和科学研究领域中的应用进行综述。     

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?%).     

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.     

实时荧光PCR方法在食品真伪辨别中的应用

实时荧光PCR技术以其特异性、灵敏性、简便、快速的优点,广泛应用于食品鉴伪检测。本文阐述了实时荧光PCR的原理以及其在肉制品、高价值食品、乳制品、果汁、水产品和油脂鉴伪检测方面的应用情况。      

Development and validation of a real-time PCR detection method for celery in food

As from 25 November 2005 onwards, a list of ingredients with known allergenic potential has to be labeled according to Directive 2003/89/EC, including celery and products thereof. In order to provide appropriate detection methods a novel real-time polymerase chain reaction (PCR) system for the specific and sensitive detection of DNA from celery (Apium graveolens) was developed and validated. Specificity was confirmed by testing DNA derived from more than 50 food relevant organisms. Sensitivity was demonstrated on the basis of a calibration curve plotting the corresponding Ct-values against DNA amounts ranging from 1 to 1000 copies. Due to the lack of certified reference material the applicability of the method was assessed by analysis of sausages spiked with defined amounts of grounded celery seed. The limit of detection (LOD) examined exemplarily for emulsion-type sausages was 5–10 mg/kg. Analysis of celery-containing commercial products demonstrated the performance potential and limitations of the new real-time PCR system.     

实时定量荧光PCR快速鉴定食品中单核细胞增生李斯特氏菌

目的建立实时定量荧光PCR法(real-time PCR)快速鉴定食品中的单核细胞增生李斯特氏菌(Listeria monocytogenes,LM)。方法选取2016年国家食品风险监测样本134例与模拟灭活LM样本10例,采用GB/T4789.30-2010与real-time PCR方法同步检测单核细胞增生李斯特菌。结果共检测食品134份,包括肉制品、水产品、快餐和即食食品等。共检出8株LM,检出率为5.97%。以GB/T4789.30-2010为金标准判断,real-time PCR方法检测样本中LM的灵敏度与特异度均达到100%。模拟灭活LM样本real-time PCR方法检出率为100%,标准法检出率为0%。结论本方法可以简化实验程序,减少工作量,节约检测试剂,为可能发生的食物中毒尽早提供实验依据。     

A real-time PCR method for the detection and quantification of lupin flour in wheat flour-based matrices

Lupin flour is growingly being used in bakery products, mainly as a soybean protein substitute. The aim of the present work was to detect and quantify the presence of lupin flour in wheat-based foods using a newly set up qPCR system based on SYBR green. Although DNA sequence information for lupin is scarce, it has been possible to design a primer pair highly specific for the target gene and devoid of any primer-dimers amplification capacity. Lupin flour revealed to be a difficult matrix, since large amounts of compounds tend to co-purify with DNA, even adopting well established extraction protocols. Nonetheless, the primers used allowed to reach high PCR efficiencies and did not show any cross-reactivity with DNAs extracted from various plant and animal foods. The sensitivity achieved was 7 pg of lupin DNA, corresponding to a percentage of less than 0.1% of lupin flour in the foods.     

Development of real-time PCR assays for the detection of lupin residues in food products

Lupin is a legume from the Leguminosae family that is used, amongst other, for human nutrition. In Europe, lupin is used as a substitute for soy in bakery and dietary products and recently its consumption has increased significantly. Unfortunately lupin is known to trigger allergic reactions in sensitised individuals and therefore its use in food products requires a mandatory declaration on the label in accordance with Directive 2007/68/EC. To protect the allergic consumer the availability of detection methods for the identification of lupin in food products is required. Here we present the development of two real-time polymerase chain reaction (PCR) methods that allow the detection of lupin-specific DNA as a marker for the presence of this allergenic ingredient in food products. Genomic DNA sequences coding for conglutin genes were chosen as targets for the detection of lupin. One primer set and probe was designed for the amplification of a 153 bp fragment of α-conglutin; another primer set and probe was designed for the detection of a 150 bp δ-conglutin amplicon. Lupin at a level of 10 mg/kg food matrix could be detected in cookies baked from a lupin containing dough using the α-conglutin method. Since lupin is used in bakery products the effects exerted by heat treatments on lupin detection by real-time PCR have been investigated. Enzyme-linked immunosorbent assay (ELISA) analyses were performed in parallel to compare the detection of lupin DNA with that of lupin protein in market products. Qualitative ELISA results confirm results obtained by the real-time PCR methods targeting α- and δ-conglutin.