Relative quantification of walnuts and hazelnuts in bakery products using real-time polymerase chain reaction

Two versions of real-time polymerase chain reaction (PCR) for relative quantification of walnuts and hazelnuts in bakery products were developed. The first method is a duplex real-time PCR with 5′-nuclease (TaqMan) probes labelled with FAM and JOE for walnuts and hazelnuts, respectively. The second method uses two real-time PCR assays for walnuts and hazelnuts with TaqMan probes labelled with FAM, carried out in separate microtubes, with normalisation of the results achieved by adding wheat as an internal standard. The internal standard is quantified in a duplex format using the TaqMan probe labelled with JOE. Both methods produced linear calibration curves (r² = 0.9813 for the former method, r² = 0.9992 for the latter) in the range 10–90% (w/w) for walnut–hazelnut mixtures. Almost identical calibration curves were obtained also for walnut–hazelnut mixtures diluted with inert matrix (oat flakes; the inert matrix accounting for 80 and 96% (w/w), respectively). Practical applicability of the developed methods was demonstrated on bakery products from the market. The developed methods will be useful for food control laboratories to facilitate authentication of bakery products with nut filling.     

Quantification of Hazelnut Content in Fillings of Cakes and Wafers Using Real-Time Polymerase Chain Reaction

A method for quantification of hazelnuts in fillings of cakes and wafers, using macadamia nuts as an internal standard material, was developed and evaluated. The method was based on quantitative adaptation of the previously developed real-time PCR for identification of hazelnuts. Calibration lines were constructed for two types of fillings, using series of model mixtures. The model mixtures for calibration of cake fillings consisted of hazelnuts and walnuts; the model mixtures for calibration of wafer fillings (pastes) consisted of hazelnuts and peanuts. Linear calibration lines were obtained for both types of matrix, and practical applicability of the developed method was demonstrated on food products from the market. The developed method may be useful for food control laboratories to facilitate analysis of food products with hazelnut filling.     

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.     

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 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.     

Application of a qualitative and quantitative real-time polymerase chain reaction method for detecting genetically modified papaya line 55-1 in papaya products

Genetically modified (GM) papaya (Carica papaya L.) line 55-1 (55-1), which is resistant to papaya ringspot virus infection, has been marketed internationally. Many countries have mandatory labeling regulations for GM foods, and there is a need for specific methods for detecting 55-1. Here, an event- and construct-specific real-time polymerase chain reaction (PCR) method was developed for detecting 55-1 in papaya products. Quantitative detection was possible for fresh papaya fruit up to dilutions of 0.001% and 0.01% (weight per weight [w/w]) for homozygous SunUp and heterozygous Rainbow cultivars, respectively, in non-GM papaya. The limit of detection and quantification was as low as 250 copies of the haploid genome according to a standard reference plasmid. The method was applicable to qualitative detection of 55-1 in eight types of processed products (canned papaya, pickled papaya, dried fruit, papaya-leaf tea, jam, puree, juice, and frozen dessert) containing papaya as a main ingredient.     

超高效液相色谱法检测焙烤食品馅料中的 二氧化硫脲

目的 建立馅料中二氧化硫脲的超高效液相色谱测定方法。方法 用0.05%乙酸水溶液提取,样品提取液经过离心,乙腈稀释过滤膜过滤后,以HILIC色谱柱为分析柱分离,采用超高效液相色谱-二极管阵列检测器(UPLC - PDA)检测,以乙腈和水为流动相,梯度洗脱,检测波长为284 nm。采用外标法定量。结果 二氧化硫脲的线性范围为0.4 ~ 20 mg/L,相关系数不小于0.999,空白样品的加标回收的回收率为80.6% ~ 102.2%,相对标准偏差为2.6% ~ 5.0%。结论 该方法简便、快速、准确、重现性好,适用于馅料中二氧化硫脲的检测。     

Real-time multiplex PCR: An accurate method for the detection and quantification of 35S-CaMV promoter in genetically modified maize-containing food

A very sensitive and new real-time multiplex PCR method for the quantification of genetically modified (GM) maize crops in food materials was developed and validated for an ABI Prism 7700 Sequence Detection System. In the assay described, fluorescence-labelled TaqMan probes were chosen to detect the amplified DNA fragments during PCR. In this multiplex approach, maize-specific DNA (zein) and 35S-CaMV promoter-specific DNA fragments are amplified in the same tube. The method was tested for the detection and quantification of the four maize events that are approved in Europe and contain the 35S-CaMV promoter: Bt11, Bt176, Mon810 and T25 maize. Quantification was based on a standard curve prepared from certified maize flour reference material prepared by the Institute for Reference Materials and Measurements. Quantification within the range of the standard curve (0.05-1% GM maize) and up to 100% was possible. Repeatability of the method for each GM maize event was determined; coefficients of variations ranged from 28-40%. In addition, three internal Nestlé laboratories successfully applied this method and comparable results were obtained.     

Validation of end-point and real-time PCR methods for the rapid detection of soy allergen in processed products

This work describes the development and validation of two PCR methods, end-point and real-time PCR, for the detection of soy protein in a wide rage of foodstuffs. These techniques are reliable and sensitive, allowing detection of trace amounts of soybean in processed products. TaqMan real-time PCR was the simpler and more rapid process, with a higher potential for automation and, therefore, currently the most suitable screening method. To verify correct operation of the proposed methodology, ELISA was used for quantitative determination of soy protein. In addition, 35 meat, fish and bakery processed products, which could potentially contain soy but was not declared on the label, were tested for the presence of soy DNA using the proposed methods. The methodologies will be valuable in issues regarding the presence of soy protein in processed products, especially in verifying labelling and security regulations to protect consumer's rights.     

A statistical approach for evaluation of PCR results to improve the practical limit of quantification (LOQ) of GMO analyses (SIMQUANT)

The predominant approach for quantification of genetically modified organisms (GMO) is the application of quantitative real-time PCR. However, for a large number of processed food and feed products, this approach is unsuitable, because they contain low amounts (mass) of amplifiable DNA. Here we present a novel approach, “Single molecule quantification” (SIMQUANT) for GMO quantification of samples with extremely low amounts of DNA. The approach is based on statistics and application of multiple qualitative parallel PCRs. Here the qualitative PCRs were done using real-time PCR setup, but this is not a requirement. The difference is that the quantitative real-time PCR requires that the target copy number exceeds the absolute limit of quantification (LOQ_abs) and provides quantity estimates by extrapolation from a linear regressional relationship between an observed cycle threshold (Ct) value and copy numbers, while with SIMQUANT the template DNA typically contains very few, e.g., one target copy per PCR volume and the quantity is estimated on the basis of observed ratio between positive and negative individual PCRs. The components of this analysis are the numbers of test samples, the size of each sample and the outcome in number and relative ratio of positive and negative test results. The approach results in a statistical estimate of the relative GM concentration based on the probability that one or more amplifiable GM template copies are present in discrete volumes. Thus, the approach is based on the ratio of discrete volumes without or with one or more PCR-amplifiable GM target copies. The approach described here can be used reliably with more than a 100-fold improvement of the practical LOQ (LOQ_pract) compared to real-time quantitative PCR based on standard curves.     

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.     

Survey of undeclared soy allergen levels in the most frequently recalled food categories with or without precautionary labelling

A comprehensive study was designed to determine the frequency and levels of soy allergen in packaged bakery and snack food products. A representative sample of products with no soy allergen disclosed on the label was analysed using two widely used enzyme-linked immunosorbent assay (ELISA) methods. Samples were chosen that either had no soy identified on the product label or which had a soy precautionary statement. Among 558 bakery and snack products, soy protein was detected in 17% of the products using the Neogen (NE) kit and 11% of the products using the Elisa Systems (ES) kit. The disagreement rates between kits were 8.8% for bakery products and 3.3% for snack products. Overall soy protein was detected at higher frequency in bakery products than in snack foods. Among 284 bakery samples, soy protein was detected in 25% of the samples with no precautionary statement and 19% of the samples which had a precautionary statement. Among 274 snack samples, soy protein was detected in 11% of the samples with no precautionary statement and 9% of the samples which had a precautionary statement. The sample repeatability was at an acceptable level (< 9%) for each method and food commodity. The reproducibility between kits was 23% for bakery foods and 36% for snack foods. None of the bakery (21) and snack (6) products without precautionary labelling (measured level > 5 ppm) had a higher level of soy protein per serving compared with the eliciting dose₁₀ (ED₁₀) of 10.6 mg for soy allergic patients. But the level of soy protein per serving may be clinically relevant to a subpopulation of soy allergic patients if a more stringent eliciting dose is applied. These findings emphasise that suitable detection methodologies and references doses are crucial for labelling accuracy and the safety of soy allergic consumers.     

Quantitative detection of species-specific DNA in feedstuffs and fish meals

A sensitive and rapid method for the quantitative detection of bovine-, ovine-, swine-, and chicken-specific mitochondrial DNA sequences based on real-time PCR has been developed. The specificity of the primers and probes for real-time PCR has been tested using DNA samples of other vertebrate species that may also be present in rendered products. The quantitative detection was performed with dual-labeled probes (TaqMan) using absolute quantification with external standards of single species meat-and-bone meals. This method facilitates the detection of 0.01% of the target species-derived material in concentrate feed mixtures and fish meals.     

凝胶渗透色谱净化-气相色谱-质谱-内标法 测定焙烤食品中富马酸二甲酯

目的 建立凝胶渗透色谱净化-气相色谱-质谱-内标法测定焙烤食品中富马酸二甲酯残留量的方法。方法 试样经乙酸乙酯超声提取,经凝胶渗透色谱(GPC)净化,采用选择离子监测模式(SIM)进行气相色谱-质谱(GC-MS)测定,采用丁二酸二甲酯作为内标,以内标法定量。结果 富马酸二甲酯在0.02 ~ 5 mg/L 内呈良好的线性关系,相关系数(r)大于0.9999;0.1、0.2和1.0 mg/kg水平的空白糕点加标回收实验的回收率为92.0% ~ 107 %,相对标准偏差(RSD)为3.2% ~ 5.7 % (n=6);方法的检出限(LOD)为0.012 mg/kg,定量限(LOQ)为0.040 mg/kg。结论 该方法准确,灵敏度高,适用于糕点等焙烤食品中富马酸二甲酯残留水平测定。     

Comparative studies of a real-time PCR method and three enzyme-linked immunosorbent assays for the detection of central nervous system tissues in meat products

The removal of certain central nervous system (CNS) tissues (part of the bovine spongiform encephalopathy risk material) from the food chain is one of the highest priority tasks associated with avoiding contamination of the human food chain with the agent of bovine spongiform encephalopathy. A recently developed real-time PCR assay and three commercially available enzyme-linked immunosorbent assays (ELISAs) for the detection of CNS tissues in minced meat and three types of heat-treated sausages were evaluated. Bovine brain was used for spiking of internal reference material, and its detectability was examined during storage times of 12 months (for frozen minced meat and liver sausage) and 24 months (for sausages treated with medium and high heat). The real-time PCR method and both ELISA kits detected 0.1% CNS tissue in frozen minced meat and 0.1 or 1% CNS tissue in heat-treated meat products. The detectability of the amplified mRNA target region with the PCR assay was similar to the detectability of antigen by the ELISAs. Because the real-time PCR method also can be used to distinguish cattle, ovine, and caprine CNS tissues from porcine CNS tissues, it seems to be suitable as a routine diagnostic test for the sensitive and specific detection of CNS tissues in meat and meat products.     

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.     

A novel PCR method for quantification of buckwheat by using a unique internal standard material

A novel quantitative and specific method for detection of buckwheat, a known food allergen, in diverse food materials was developed by using a unique internal standard to compensate for the variability in DNA extraction and amplification efficiencies. The method was based on a real-time PCR targeting the internal transcribed spacer region of Fagopyrum spp. and was designed to detect both cultivated and wild buckwheat, because wild buckwheat might be potentially allergenic. As the internal standard material, ground seeds of statice (Limonium sinuatum) were added to food samples prior to DNA extraction, and the amount of statice DNA measured by real-time PCR was used to standardize the buckwheat content. Statice, an ornamental plant, was chosen as the internal standard material because it was readily available and was inferred to be least likely to be commingled in foods. The specificity of the PCR system was tested against commonly used food materials of plant origin. Quantitative results expressed in buckwheat protein concentrations (mean +/- standard deviation) for various food samples prepared to contain 10 ppm (wt/wt) of buckwheat flour (corresponding to 1.2-microg/g [ppm] buckwheat protein) ranged from 0.7 +/- 0.2 (rice) to 0.9 +/- 0.4 (wheat) and for 100-ppm (wt/wt) samples (12-microg/g [ppm] buckwheat protein) from 7.7 +/- 1.0 (pepper) to 9.8 +/- 0.5 (wheat) microg/g (ppm). The method's accuracy, sensitivity, and specificity were considered sufficient for detection of buckwheat contamination at the level required for compliance with the Japanese Food Allergen Labeling Regulation.     

Development of a quantitative GC-FID method for the determination of stearoyl-lactylates (E481/482) in foods

Sodium and calcium salts of stearoyl-lactylates (SLs) are food emulsifiers especially used in bread and bakery products to improve texture. They should be used at the lowest level at which the desired technological effect is achieved in a specific food category and at amounts not exceeding the maximums set by European Commission Regulation No. 1129/2011. In order to be able to evaluate whether these emulsifiers are used correctly but also to evaluate whether the commercial additive formulations comply with legislation, a quantitative GC-FID method was developed. An internal standard (nonadecanoyl-1-lactylate) was synthesized in-house and pure ester standards were isolated from commercial additive formulations. The method showed a limit of detection of 0.04 and a limit of quantification of 0.12 mg esters ml^?1. The commercial additive formulations analysed proved to be complex mixtures of free lactic and fatty acids together with only 50–60% esters. Besides SLs important amounts of palmitoyl-lactylates were present. Different food matrices (with low- and high-fat contents) were spiked with commercial SL formulations and recoveries ranged between 85% and 109%. Determination of SLs in commercial foods (such as bakery and bread) indicated that pre-treatment with amylase was essential to determine accurately the SL content due to the interaction of SL with the amylose.     

Development of a quantitative real-time PCR assay for the detection of Aspergillus carbonarius in grapes

Aspergillus carbonarius is the main species responsible for the production of ochratoxin A (OTA) in wine grapes. To monitor and quantify A. carbonarious in grapes, a quantitative real-time PCR assay was developed as a possible tool for predicting the potential ochratoxigenic risk. DNA extraction from grape berries was performed by using conventional extraction and clean up through EZNA Hi-bond spin columns. A TaqMan probe was used to quantify A. carbonarius genomic DNA in grape berries samples. An exogenous internal positive control was used to overcome DNA recovery losses due to matrix inhibition. The quantification of fungal genomic DNA in naturally contaminated grape was performed using the TaqMan signal versus spectrophotometrically measured DNA quantities (Log10) calibration curve with a linearity range from 50 to 5 x 10(-4) ng of DNA. A positive correlation (R2=0.92) was found between A. carbonarious DNA content and OTA concentration in naturally contaminated grape samples. This is the first application of TaqMan real-time PCR for identifying and quantifying A. carbonarius genomic DNA occurring in grapes. The rapid DNA extraction method for grapes, together with the commercial availability of reagents and instrumentation, allows to perform a remarkable number of reproducible assays (96-well format) in less than 4 h.     

Integrated structure and event-specific real-time detection of transgenic <Emphasis Type="Italic">cry1Ac/SCK</Emphasis> rice Kefeng 6

Transgenic rice Kefeng 6 is a transformation event containing two insect-resistant genes, cry1Ac and SCK (modified CpTI gene) in China. In order to monitor the probable release of Kefeng 6 in the future and execute the labeling requirements, it is necessary to develop a rapid and reliable detection method. In this study, both the 5′ and 3′-junction sequences spanning the plant DNA and the integrated gene construct of the rice event Kefeng 6 were isolated by genome walking and long-distance PCR (LD-PCR), successively. Multiple copies of truncated SCK gene and cry1Ac gene were found to integrate into the host rice genome. The event-specific real-time detection method for Kefeng 6 event based on its 5′-junction sequence was established using one plasmid molecule pMD-KF6 containing both 5′-junction sequence and rice endogenous gene gos9 sequence as the reference material (RM) with an absolute limit of quantification (LOQa) around 10 template copies. Thereafter, three different transgenic amounts of w/w mixed samples (5, 1, and 0.5%, respectively) were quantified to assess the performance characteristics of the established real-time PCR method. The accuracy expressed as bias deviated from the 4.00–26.00%, the precision expressed as standard deviation (SD) and relative standard deviation (RSD) deviated from 0.03–0.19 and 3.42–4.76%, respectively. Based on the earlier results, we concluded that the qualitative and quantitative PCR assays were reliable and accurate for Kefeng 6 measurement, and the reference plasmid pMD-KF6 could be a good substitute for the reference material for Kefeng 6 quantification.