Development and validation of a duplex real-time PCR method for the simultaneous detection of celery and white mustard in food

The developed duplex real-time PCR method allows the simultaneous detection of traces of potentially allergenic white mustard (Sinapis alba) and celery roots (Apium graveolens var. rapaceum), celery stalks (A. g. var. dulce) and leaf celery (A. g. var. secalinum). The duplex assay does not show any cross-reactivity with 64 different biological species, including various members of the Brassicaceae and Apiaceae family. In raw model sausages spiked with white mustard and celery roots, the LOD was found to be 0.001% white mustard and 0.005% celery. In model sausages brewed at 75–78 °C for 15 min the LOD was found to be 0.005% white mustard and 0.005% celery. The duplex real-time PCR assay was applied to check if commercial food products are labelled in compliance with the legal regulations.     

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 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.     

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.     

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.     

Chirale Phthalid-Aromastoffe: (3a–7a)-cis-3-Butylhexahydrophthalid-Stereoisomere in Knollensellerie (Apium graveolens L var.rapaceum)

In diethyl ether extracts from celeriac (Apium graveolens L. var.rapaceum) all four stereoisomers of (3a–7a)-cis-3-butylhexahydrophthalide were found to be present. The analyses were carried out by means of GC, enantioselective GC and GC-MS. The assignment of the relative configuration of the diastereomers3 and4 was accomplished by NOE difference spectroscopy.     

Rapid detection of Listeria monocytogenes in food using culture enrichment combined with real-time PCR

A rapid method for the detection of Listeria monocytogenes in foods combining culture enrichment and real-time PCR was compared to the ISO 11290-1 standard method. The culture enrichment component of the rapid method is based on the ISO standard and includes 24 h incubation in half-Fraser broth, 4 h incubation in Fraser broth followed by DNA extraction and real-time PCR detection of the ssrA gene of L. monocytogenes. An internal amplification control, which is co-amplified with the same primers as the L. monocytogenes DNA, was also included in the assay. The method has a limit of detection of 1–5 CFU/25 g food sample and can be performed in 2 working days compared to up to 7 days for the ISO standard. A variety of food samples from retail outlets and food processing plants (n = 175) and controls (n = 31) were tested using rapid and conventional methods. The rapid method was 99.44% specific, 96.15% sensitive and 99.03% accurate when compared to the standard method. This method has the potential to be used as an alternative to the standard method for food quality assurance providing rapid detection of L. monocytogenes in food.     

Detection of genetically modified rice: a construct-specific real-time PCR method based on DNA sequences from transgenic Bt rice

Genetically modified rice varieties developed in China are close to approval for agricultural cultivation and production. However, so far no method has been reported for specific detection of transgenic varieties of this crop. In the present study, rice seeds assumed to consist of field-tested Bt rice (‘Anti-pest Shanyou 63’ and ‘Anti-pest Jinyou 63’) were used as reference material to determine transgenic DNA sequences. The transition between the cryIA(b) and cryIA(c) fusion gene and the nopaline synthase terminator (nos) sequence was used to develop a construct-specific real-time PCR based detection method. This Bt rice specific detection system was combined with a recently published quantitative real-time PCR method for the rice-specific (Oryza sativa L.) reference gene gos9. The complete PCR assay for detection of transgenic Bt rice was in-house validated and the limit of quantification was found to be below 0.1% Bt rice relative to the rice content. Application of the PCR assay should allow more precise detection of transgenic rice varieties in imported food products which are so far not approved in the EU.     

ITS-based detection and quantification of Alternaria spp. in raw and processed vegetables by real-time quantitative PCR

A TaqMan real-time polymerase chain reaction (PCR) method was developed for specific detection of Alternaria spp. in foodstuffs. The method uses Alternaria-specific primers and probe targeting the internal transcribed spacer regions ITS1 and ITS2 of the rRNA gene, and a positive amplification control based on 18S rRNA gene. The applicability of the real-time PCR protocol was assessed through analysis of 190 commercial food samples, including 80 fresh fruit and vegetable samples and 110 processed foodstuffs. The assay demonstrated the presence of Alternaria spp. DNA in 46 out of the 80 raw samples (57.5%) and in 66 out of the 110 processed samples (60%), enabling quantitative detection of Alternaria spp. DNA at levels as low as 1 CFU/g. The estimated Alternaria counts obtained by real-time PCR showed a good relationship (R² = 0.9006, P < 0.01) with the Alternaria counts obtained by plating on Potato Carrot Agar (PCA). The developed real-time PCR assay provides a useful tool for early detection of Alternaria spp. and could be applied as a quality and biosecurity marker of raw materials and final products in the fruits and vegetables processing industries.     

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.     

Causes of Decay of Fresh-cut Celery

A decay of fresh-cut celery (Apium graveolens) segments stored at < 5°C in sealed film bags began with a water soaking of the cut surfaces. Slimy moisture accumulated inside the bags. The segments water soaked completely, softened, discolored and sometimes disintegrated. Total aerobic bacterial populations isolated from decayed segments ranged from log₁₀ 7.0–7.7 CFU/g tissue weight. The predominant bacteria, identified by fatty acid analysis as Pseudomonas fluorescens and P. marginalis, caused water soaking, soft rot, and discoloration in freshly inoculated celery tissues stored at 5 or 25°C. Leuconostoc mesenteroides was also isolated and may have been responsible for slime production.     

Potential Inoculum Sources for Decay of Fresh-cut Celery

Bacteria responsible for decay of precut celery (Apium graveolens) appear to be residents on the plants at harvest. Bacterial genera isolated from equipment in a fresh celery packinghouse were similar to those found on plants sampled at the packinghouse entrance. Population densities and array of genera were highest on unwashed plants, whereas certain genera were no longer detected on the washed plants. Fluorescent Pseudomonas spp. predominated, but strains of Pantoea agglomerans (=Erwinia herbicola), Enterobacter agglomerans, Aeromonas, Arthrobacter, and Aureobacterium were frequently isolated. Members of the coliform group such as Citrobacter, Klebsiella, and Escherichia were rare.     

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.     

A filtration-based real-time PCR method for the quantitative detection of viable Salmonella enterica and Listeria monocytogenes in food samples

We developed a novel filtration-based method that can eliminate dead or severally damaged Salmonella enterica and Listeria monocytogenes in food samples. This new method can recover all viable bacteria in less than 30 min, and can be coupled with a subsequent bacterial DNA extraction and real-time PCR. No statically significant differences (p < 0.01) were found between real-time PCR results obtained separately from S. enterica and L. monocytogenes when different ratios of living and dead cells were used. The analytical sensitivity in both cases was 1 genome equivalent (GE), and the quantification was linear (R² > 0.9969) over a 5-log dynamic range with PCR efficiencies >0.9754. When compared with the standard microbiological methods for the detection of these foodborne pathogens, the relative accuracy was excellent ranging from 95.72% to 104.48%. Finally, we applied the pre-treatment method to the direct detection of viable forms of these foodborne pathogens in food samples using yogurt as a model, the results being similar to those obtained using pure cultures.     

Water Vapor Resistance of Red Delicious Apples and Celery Sticks Coated with Edible Caseinate-Acetylated Monoglyceride Films

An optimal caseinate-acetylated monoglyceride edible coating consisting of 1.5% Alanate-310 (a calcium caseinate) and 1.5% Myvacet-5-07 (an acetylated monoglyceride) produced a reduction (75%) in moisture loss from celery (Apium graveolens) sticks. Similar edible coatings on apple (Malus domestica) fruits produced higher water vapor resistance. However, since apples have a natural high resistance to water vapor, edible coatings did not decrease water loss from whole fruit. Edible coatings did not modify respiration or ethylene production by apples or celery sticks. Quality of minimally processed fruits and vegetables that are susceptible to water loss could be improved with edible coatings, but quality of commodities with high water vapor resistance would not.     

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.     

Effect of enzymes on extraction of volatiles from celery seeds

Celery (Apium graveolens L.), belonging to the family Apiaceae, is widely used as a spice, in perfumery and pharmaceutical applications. It is reported to possess several nutraceutical attributes, such as anticoagulation activity of blood plasma and prevention of cardiovascular diseases. Effects of various enzymes on the extraction of volatile oil of celery are reported in the present study. The oil yield, after cellulase, pectinase, protease and viscozyme pretreatment, was in the range 2.2–2.3% as against 1.8% in a control sample, by steam distillation. Profiling of the celery oil thus obtained by GC–MS showed that limonene, the major terpene, increased from 63% to 82% with enzyme treatment. The other major flavour compounds identified were β-selinene (16–17%), butyl phthalide and sedanolide. The study demonstrated that enzymes facilitated the extraction of celery oil with increase in oil yield with little change in either flavour profile or physicochemical properties of the oil.