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 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.     

Development and validation of a novel real-time PCR method for the detection of celery (Apium graveolens) in food

The paper presents a novel real-time PCR method allowing the detection of traces of celery (Apium graveolens) in complex food matrices. The method is based on the amplification of a sequence of the gene coding for the Apium graveolens NADPH-dependent mannose-6-phosphate reductase. It allows the detection of three commonly used celery varieties, celery roots (Apium graveolens var. rapaceum), celery stalks (Apium graveolens var. dulce) and leaf celery (Apium graveolens var. secalinum) and does not show any cross-reactivity with 64 biological species, including ten members of the Apiaceae family. The limit of detection, determined by analysing serially diluted celery extracts, is 10 pg celery DNA for all three celery varieties. In spiked model sausages, the LOD is 0.005% celery. The real-time PCR method was applied to 26 commercial food products. Celery DNA was found in one out of ten samples without any information about the presence of celery.     

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.     

One-step multiplex PCR method for the determination of pecan and Brazil nut allergens in food products

BACKGROUND: A one‐step polymerase chain reaction (PCR) method for the simultaneous detection of the major allergens of pecan and Brazil nuts was developed. Primer pairs for the amplification of partial sequences of genes encoding the allergens were designed and tested for their specificity on a range of food components. RESULTS: The targeted amplicon size was 173 bp of Ber e 1 gene of Brazil nuts and 72 bp of vicilin‐like seed storage protein gene in pecan nuts. The primer pair detecting the noncoding region of the chloroplast DNA was used as the internal control of amplification. The intrinsic detection limit of the PCR method was 100 pg mL^?1 pecan or Brazil nuts DNA. The practical detection limit was 0.1% w/w (1 g kg^?1). The method was applied for the investigation of 63 samples with the declaration of pecans, Brazil nuts, other different nut species or nuts generally. In 15 food samples pecans and Brazil nuts allergens were identified in the conformity with the food declaration. CONCLUSION: The presented multiplex PCR method is specific enough and can be used as a fast approach for the detection of major allergens of pecan or Brazil nuts in food. Copyright © 2011 Society of Chemical Industry     

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.     

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.     

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.     

In-house validation of a real-time PCR method for rapid detection of<Emphasis Type="Italic"> Salmonella</Emphasis> ssp. in food products

A real-time polymerase chain reaction (PCR) method for Salmonella ssp. detection in food samples has been developed and validated in-house. The specificity of the assay was confirmed by tests with 295 different Salmonella strains, including four strains of Salmonella bongori. When tested with extracted Salmonella DNA the lowest detected amount was found to be 5 fg, which is equivalent to approximately one genome copy. The detection limit was further determined by artificial contamination of minced meat with S. Typhimurium cells and of pastry with S. enteritidis using the most probable number approach for cell dose dilutions. It was calculated that even one colony forming unit of Salmonella was still detectable in 25 g food after enrichment culture for 18 h. An additional PCR system for internal positive control, which was included in each reaction and detected in parallel via another reporter fluorescence dye, has no negative impact on the sensitivity of the assay. The method was evaluated with 1,293 naturally contaminated food samples and compared to the conventional cultural method. Of 55 positive PCR samples, 45 were confirmed by the cultural method. The statistical comparison revealed a correlation of 99.2% for specificity, of 100% for sensitivity and of 99.2% for trueness. The results of the comparative analysis and the advantages of the real-time PCR method for detection of Salmonella ssp. under routine laboratory conditions are discussed.     

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.     

A PCR assay to detect trace amounts of soybean in meat sausages

Soybean proteins are the most widely used source vegetable proteins in the meat industry because of several interesting characteristics. As soybean is included in the group of ingredients potentially allergenic, if not declared, it can be considered a hidden allergen, representing a potential risk to sensitised individuals. The aim of this work was to optimise and apply DNA‐based techniques for soybean detection in meat products, as alternative to the currently used protein‐based methods. The optimised polymerase chain reaction (PCR) protocol targeting the soybean lectin gene enabled the detection of the addition of 0.1% and 0.5% of hydrated textured protein, which corresponded to 0.01% and 0.06% (w/w) of soybean protein in unprocessed and heat‐processed pork meats, respectively. The established PCR technique, when applied to commercial meat sausages (eighteen samples), confirmed the presence of soybean declared in nine samples and indicated the presence of soybean in four samples with no labelled information about soybean. Additionally, the event‐specific PCR detection of Roundup Ready^® soybean was also performed, enabling the detection of transgenic DNA in three samples.     

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.     

Detection of pea in food by real-time polymerase chain reaction (PCR)

A qualitative 5′-nuclease real-time PCR-based method for the detection of pea (Pisum sativum) in food is described. The method consists of DNA isolation by chaotropic solid phase extraction and the subsequent PCR with pea-specific primers and a TaqMan fluorescent probe. The primers and the probe are oriented to the chloroplast DNA intron located between trnL and trnF exons encoding for tRNA. The analytical parameters of the method were inclusivity 100%, exclusivity 100% and the detection limit of 0.11±0.07 ng of pea DNA corresponding to 12±7 diploid pea genome copies. Using a set of model meat patés with defined pea contents, a matrix-related detection limit of 0.05% was determined and a linear calibration line was constructed. The presented analytical method was useful for qualitative detection or semiquantitative determination of pea in food products. The method was relatively fast because the analysis could be performed in one working day.     

Development of an event-specific detection method for genetically modified rice Kefeng?6 by quantitative real-time PCR

The genetically modified (GM) rice Kefeng 6 has gained resistance against several rice pests by inserting the cpti and cry1Ac genes. As this transgenic line is not approved for import, processing and cultivation in the European Union (EU), sensitive and specific detection methods need to be available to monitor any illegal presence of Kefeng 6 in food products within the EU. The aim of this study was to develop and validate an event-specific detection method by means of quantitative real-time PCR (qPCR) for the detection of Kefeng 6 in foodstuff. A primer pair and hydrolysis probe were designed according to the right border junction sequence of the transgene. The qPCR assay was validated according to the ENGL/EURL-GMFF guidelines for GMO testing and is presented according to the MIQE guidelines. The in-house validation process resulted in a limit of detection of 5 DNA copies of the transgene with confidence intervals (95 %) between 0.07 and 0.52, a PCR efficiency of 105 % and a correlation coefficient (R ²) value of 0.9997. The specificity of the assay was tested by end-point PCR, gel electrophoresis and subsequent sequencing of the PCR products. By testing DNA of several GM and non-GM crops, cross reactivity of the assay was not observed. Further, 35 food products were analyzed for the presence of Kefeng 6 by means of the event-specific detection method. For 9 out of 35 samples, PCR products for Kefeng 6 DNA were observed.     

Detection of pig derivatives in food products for halal authentication by polymerase chain reaction –restriction fragment length polymorphism

A method for detection of the presence of pig derivatives in three types of food products—sausages and casings, bread and biscuits—using polymerase chain reaction–restriction fragment length polymorphism (PCR‐RFLP) analysis of a conserved region in the mitochondrial (mt) cytochrome b (cyt b) gene was developed. Genomic DNA of sausages and casings, bread and biscuits were extracted. The genomic DNA from the food products were found to be of good quality for the sausages and produced clear PCR products on the amplification of the mt cyt b gene of approximately 360 base pairs (bp). However, no genomic DNA was detected from the casing samples and poor quality of genomic DNA was extracted from bread and biscuits. No amplification of mt cyt b gene was produced from bread and biscuit samples. To differentiate between samples, the amplified PCR products were digested with restriction enzyme (RE) BsaJI, resulting in species‐specific RFLP. The cyt b PCR‐RFLP species identification assay gave excellent results for detection of pork adulteration in food products and is a potentially reliable technique to avoid species adulteration or fraudulent species substitution for halal authentication. Copyright © 2006 Society of Chemical Industry     

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 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