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 (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.     

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     

Fatty acids profile of oil from nine varieties of Macadamia nut

The fatty acids profile and lipids content of the Macadamia integrifolia and Macadamia tetraphylla varieties and interspecies hybrids, M. integrifolia and M. tetraphylla were analyzed. Total lipids content ranged from 70.9 to 79.7 g of oil per 100 g^?1 dry-solids. Gas chromatography/mass spectrophotometry identified 20 fatty acids, the most abundant of which were oleic acid (40 to 51%), palmitoleic acid (24 to 36%), and palmitic acid (8.4 to 13.1%). Fatty acids concentration differed between varieties and hybrids (Multivariate analysis of variance, F = 13.89, p < 0.0001). A ratio of monounsaturated fatty acids:saturated fatty acid values ranged from 2.8 to 4.6. The Huatusco variety (M. integrifolia and M. tetraphylla) exhibited the highest percentage of lipids (79.7 g of oil per 100 g^?1 DS) and the highest monounsaturated fatty acids:saturated fatty acid (4.6), thus highlighted its potential applications within the food industry. The Cate variety (M. integrifolia and M. tetraphylla) had the lowest monounsaturated fatty acids:saturated fatty acid, suggesting potential uses in the cosmetics industry.     

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.     

Moisture characteristics of macadamia nuts in relation to storage

The moisture content/relative humidity characteristics of nuts from two species of Macadamia have been examined. For Macadamia tetraphylla the whole nut moisture content corresponding to a relative humidity (r.h.) of 70%, accepted as the maximum for safe storage, was 11.8%. The moisture content can be determined with an electrical moisture meter by calibrating against standard laboratory procedures. The sorption isotherm of M, integrifolia appeared somewhat different.     

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.     

Validation of a Duplex Real-Time PCR for the Detection of Salmonella spp. in Different Food Products

A 5′ nuclease duplex real-time polymerase chain reaction (PCR) assay was developed and validated with various food products for the specific and fast detection of Salmonella spp. in food. The assay used previously published primers in combination with a newly developed probe targeting the invA gene. An internal amplification control, which is coamplified in a duplex PCR, was included in the assay. The analysis of 1,934 natural food samples with real-time PCR and the cultural method in parallel resulted in a relative accuracy of 100% and 99.84% respectively, depending on the enrichment procedure in which buffered peptone water and selective enrichment in Rappaport–Vassiliadis (RV) broth were employed. The duplex real-time PCR assay has proven to be a specific, sensitive and fast screening method for Salmonella spp. in food. The overall analysis time of the PCR method was approximately 28 h, in contrast to 4 to 5 days with conventional Salmonella diagnostics. The developed assay has been shown to be a reliable diagnostic tool for use in routine analysis. It has been validated thoroughly and has become an official method in Germany for the detection of Salmonella spp. in food.     

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.     

Rapid and Sensitive Detection of Staphylococcus aureus in Food Using Selective Enrichment and Real-Time PCR Targeting a New Gene Marker

Staphylococcus aureus is a bacterial pathogen considered a principal etiological agent of food poisoning. The aim of this study was to develop and evaluate a rapid and sensitive method for the detection of S. aureus in food by using selective enrichment and a new species-specific real-time polymerase chain reaction (PCR). Specific primers and a TaqMan probe targeted to specific S. aureus gene encoding for acriflavine resistance protein were designed. The real-time PCR was highly specific for S. aureus with 100% inclusivity and 100% exclusivity determined using 83 S. aureus strains and 64 non-S.-aureus strains. PCR detection limit of 6.8 × 10¹ and 3.4 × 10¹ CFU ml⁻¹ were obtained with 100% and 70% detection probability, respectively. The single selective enrichment based on the study of different enrichment conditions was selected and a lysis by boiling was used to obtain bacterial DNA. Out of 112 food samples analyzed, 61 were positive by the PCR-based method and 53 by the standard method. Out of ten food matrices artificially contaminated at a level of 10° CFU g⁻¹, ten and six were positive by the respective methods. Moreover, 10° CFU 10 g⁻¹ was detected in all ten artificially contaminated samples after a large-scale enrichment using PCR-based detection, in contrast to seven false negative by standard detection. The developed method facilitated the detection of S. aureus on the next day after the sample reception. This method can be used for S. aureus detection as a faster, highly specific, and more sensitive alternative to microbiological method with the potential for providing of improved food-processing hygiene control.     

食品过敏原澳洲坚果环介导等温扩增检测方法的建立与应用

根据澳洲坚果豌豆蛋白AMP2基因序列,利用设计软件Primer Explorer Version 4设计并筛选了食品过敏原澳洲坚果的环介导等温扩增引物,对反应体系和反应条件进行优化,建立澳洲坚果的环介导等温扩增检测方法,结果判断可采用实时荧光法和荧光染料终点显色法。对该方法进行了特异性、灵敏度、稳定性评价,结果显示：该方法能够特异性、灵敏、稳定地检测食品中的澳洲坚果成分,检测低限为0.5%。此外,对7 种市售食品样品的检测结果表明,该方法与食品标签标示的过敏原成分结果吻合率为100%,假阳性率和假阴性率均为0,在市售食品的过敏原成分检测上较商业化快速检测试纸条更加稳定可靠。     

Detection of Raw Pork Targeting Porcine-Specific Mitochondrial Cytochrome B Gene by Molecular Beacon Probe Real-Time Polymerase Chain Reaction

Pork identification in raw meat using real-time polymerase chain reaction (PCR) was developed. Total DNA from meat samples were successfully extracted and found to be of high quality and produced clear PCR products. Porcine-specific molecular beacon probe and primers that amplifies 119 bp of the cytochrome b gene fragment of swine (Sus scrofa domestica) was used. Analysis of data showed that the C _q (quantification cycle) from 10 ng/μl porcine DNA is (18.70 ± 0.12 to 19.08 ± 0.06). Meanwhile, the other samples exhibited negative result, which confirmed the specificity of the primers. The method also showed that the limit of detection of pork was 0.0001 ng. Based on the regression analysis of the standard curve, the 96% efficiency of real-time PCR was achieved with high correlation coefficient (r ² = 0.9989). Sensitivity of the assay in discriminating pork as low as 0.1% (w/w) pork in pork–beef mixtures was also obtained. Reproducibility of the assay was successfully validated by applying sample and experimental replicates in every assay being conducted. Thus, this methodology could serve as a fast and sensitive method for detection of pork for meat species verification.     

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.     

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.     

Optimal salting of macadamia (Macadamia integrifolia) nuts

Roasted macadamia (Macadamia integrifolia Storey) nuts were salted at various levels up to 19 g kg^?1 and presented to a taste panel. The results showed that the assessors could be divided into 'salt dislikers', to whom perceived quality decreased linearly with increase in salt content, and 'salt likers', to whom perceived quality increased as the salt content increased. It follows that the nuts can be produced to suit these two market segments separately, or else a single lightly salted compromise product can be offered to both groups.     

Development of an Internal Amplification Control Using Multiplex PCR for the Detection of Listeria monocytogenes in Food Products

The bacterial pathogen Listeria monocytogenes is responsible for listeriosis, a food-borne disease, which may result in severe illness and possible death. Large outbreaks of listeriosis have been associated with food products including soft cheeses and ready to eat food products. Polymerase chain reaction (PCR) is a molecular identification method for food-borne pathogens; however, a drawback of this method is that false-positive or false-negative results may occur. To validate the accuracy of the PCR as a powerful molecular tool for pathogen detection, it is important that false-negative results be distinguishable from true-negative PCR results. The aim of this study was to design and include an internal amplification control (IAC) within the PCR to coamplify with L. monocytogenes in order to identify false-negative results of L. monocytogenes from ostrich meat and camembert cheese samples. The IAC had to be incorporated into the PCR without loss of specificity and sensitivity on the detection limit of L. monocytogenes and was developed and tested for use in a multiplex PCR detection system. A region of the pUC19 plasmid was selected as the IAC for this study. The optimal concentration at which pUC19 would coamplify with L. monocytogenes was determined to be 0.001 pg/μL. Following an enrichment procedure, the minimum number of organisms detected in a spiked food sample by the PCR was 8 CFU/mL L. monocytogenes; the same detection limit was attained when the pUC19 IAC was included in the PCR. An optimal pUC19 IAC concentration increased the reliability of the PCR for food diagnostic purposes.     

<Emphasis Type="Italic">In silico</Emphasis> phylogenetic analysis of lactic acid bacteria and new primer set for identification of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> in food samples

Lactic acid bacteria (LAB) include species very closely related both physiologically and genotypically. Therefore, the identification of this bacteria group using conventional phenotypic methods is ambiguous and cumbersome. In this study, we have analyzed a recA gene fragment from 30 bacteria, including LAB and species common in the human gastrointestinal tract, aiming to evaluate the gene conservation among them and the development of primers and PCR conditions able to discriminate Lactobacillus plantarum strains from LAB closely related. The fragment with 995 bp of recA gene has grouped LAB, enterobacteria and bifidobacteria, in different clusters. A novel primer pair, LPrecAF and LPrecAR with 23 and 18 bp, respectively, has allowed the single amplification of a 108 bp fragment of L. plantarum strains contained in culture broth and fermented dairy samples. The observed detection limit for food samples and for cultures broth were 1 × 10³ and 7 × 10² CFU mL⁻¹, respectively. This approach proved to be a simple and efficient method for the identification and monitoring of L. plantarum in food, feeds, and culture broth. Moreover, the assay could be used in the studies from human or environmental microbiota.     

Quality Evaluation of Shelled and Unshelled Macadamia Nuts by Means of Near‐Infrared Spectroscopy (NIR)

The quality of shelled and unshelled macadamia nuts was assessed by means of Fourier transformed near‐infrared (FT‐NIR) spectroscopy. Shelled macadamia nuts were sorted as sound nuts; nuts infected by Ecdytolopha aurantiana and Leucopteara coffeella; and cracked nuts caused by germination. Unshelled nuts were sorted as intact nuts (<10% half nuts, 2014); half nuts (March, 2013; November, 2013); and crushed nuts (2014). Peroxide value (PV) and acidity index (AI) were determined according to AOAC. PCA‐LDA shelled macadamia nuts classification resulted in 93.2% accurate classification. PLS PV prediction model resulted in a square error of prediction (SEP) of 3.45 meq/kg, and a prediction coefficient determination value (R_p²) of 0.72. The AI PLS prediction model was better (SEP = 0.14%, R_p² = 0.80). Although adequate classification was possible (93.2%), shelled nuts must not contain live insects, therefore the classification accuracy was not satisfactory. FT‐NIR spectroscopy can be successfully used to predict PV and AI in unshelled macadamia nuts, though.