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This paper discusses the development of rapid, reliable, and accurate polymerase chain reaction (PCR) assays for detecting opium poppy (Papaver somniferum L.) in food. Endpoint, quantitative, and digital PCRs were compared based on the amplification of a newly developed DNA marker targeting the NADPH-dependent codeinone reductase (COR) gene. Designed assays were shown to be highly specific and sensitive in discriminating opium poppy from other plant species, even in heat-treated and food samples. Digital PCR was the most sensitive, with a detection limit of up to 5 copies, i.e., approximately 14 pg of target DNA per reaction. Quantitative and digital PCR further allowed the quantification of opium poppy in up to 1.5 ng and 42 pg (15 copies) of target DNA in a sample, respectively. In addition, two duplex PCRs have been developed for the simultaneous detection of opium poppy DNA and representatives of (i) the Papaveraceae family or (ii) the Plantae kingdom. Finally, all designed assays were successfully applied for analysis of 15 commercial foodstuffs; two were suspected of being adulterated. The study results have an important impact on addressing food fraud and ensuring the safety and authenticity of food products. Beyond food adulteration, the study may also have significant implications for forensics and law enforcement.

The success of polymerase chain reaction (PCR) assay depends on template deoxyribonucleic acid (DNA) being sufficient with respect to both quantity and quality. Some biological materials contain compounds which inhibit the functioning of DNA polymerase and thus need to be removed as part of the DNA extraction procedure. The aim of the present experiments was to optimise the process of DNA isolation from various types of black, fruit and herbal teas. A comparison was made between two cetyltrimethylammonium bromide (CTAB)-based protocols and two commercially available DNA purification kits. The yield and integrity of the extracted DNA were monitored both spectrophotometrically and using agarose gel electrophoresis. The presence/absence of inhibitors in the DNA preparations was checked by running quantitative real-time PCRs. The optimal protocol was deemed to be the CTAB method described in ISO 21571:2005, so this method is recommended for the routine sample analysis of tea products.

Abstract Opium poppy (Papaver somniferum L.) is a versatile plant exploited by the pharmaceutical and food industries. Unfortunately, it is also infamously known as a source of highly addictive narcotics, primarily heroin. Drug abuse has devastating consequences for users and also has many direct or indirect negative impacts on human society as a whole. Therefore, developing a molecular genetic tool for the individualization of opium poppy, raw opium or heroin samples could help in the fight against the drug trade by retrieving more information about the source of narcotics and linking isolated criminal cases. Bioinformatic analysis provided insight into the distribution, density and other characteristics of roughly 150 thousand microsatellite loci within the poppy genome and indicated underrepresentation of microsatellites with the desired attributes. Despite this fact, 27 polymorphic STR markers, divided into three multiplexed assays, were developed in this work. Internal validation confirmed species-specific amplification, showed that the optimal amount of DNA is within the range of 0.625–1.25 ng per reaction, and indicate relatively well balanced assays according to the metrics used. Moreover, the stutter ratio (mean + 3 SD 2.28–15.59%) and allele-specific stutters were described. The analysis of 187 individual samples led to the identification of 158 alleles in total, with a mean of 5.85 alleles and a range of 3–14 alleles per locus. Most of the alleles (151) were sequenced by the Sanger method, which enabled us to propose standardized nomenclature and create three allelic ladders. The OpiumPlex system discriminates most of the varieties from each other and pharmaceutical varieties from the others (culinary, dual and ornamental).

Various diseases and pests cause serious damage to vegetable crops during the growing season and after harvesting. Growers attempt to minimize losses by protecting their crops, starting with seed and seedling treatments and followed by monitoring their stands. In many cases, synthetic pesticide treatments are applied. Integrated pest management is currently being employed to minimize the impact of pesticides upon human health and the environment. Over the last few years, “smart” approaches have been developed and adopted in practice to predict, detect, and quantify phytopathogen occurrence and contamination. Our review assesses the currently available ready-to-use tools and methodologies that operate via visual estimation, the detection of proteins and DNA/RNA sequences, and the utilization of brand-new innovative approaches, highlighting the availability of solutions that can be used by growers during the process of diagnosing pathogens.

We have developed and validated post-PCR (polymerase chain reaction) high resolution melting (HRM) based assays that allow identifying the presence of two major allergenic foods - soybean and bovine milk - in food products. A new set of primers for PCR was developed for detection of the gene encoding the soybean protein lectin. The assay was validated using reference samples and used for the analysis of artificially prepared mixed samples of dairy and soy products of different matrices, as well as of real products available in the market (spray creams). The limits of detection (LODs) of the soybean (8 copies) and bovine milk (4 copies) assays were lower compared to LODs of other previously published PCR-based assays. The analysis of several commercial spray creams revealed an undeclared presence of soybean in one of the samples. The newly developed HRM assays are precise and robust alternatives for the control of food composition and falsification by competent authorities.

Seeds of Czech poppies used for culinary purposes are frequently mixed with seeds of imported low-quality poppies with higher alkaloid content and poor taste properties. That not only decreases the quality of the product on the market, but it can also represent a potential health hazard for consumers. To prevent such adulteration, the method is needed that could clearly detect the presence of the low-quality poppies in the products on the market and which can also confirm the authenticity of the genetic material for growers. For that reason, length polymorphism of Simple Sequence Repeats was evaluated in the blue and non-blue poppy varieties of Czech and foreign origin. Used markers clearly distinguished 'Czech blue poppy' cultivars from blue poppies of another origin as well as from poppies of non-blue seed colour. We conclude that used markers can be applied to a commodity verification on the market to detect/exclude the presence of other genotypes.

The transcription activities of genes somehow associated with the mycotoxin deoxynivalenol (DON) biosynthesis, namely Fusarium Tri genes, and the barley gene coding for UDP-glycosyltransferase (HvUGT13248) on different genetic backgrounds were compared. Determining the amount of the pathogen DNA was used as a useful tool for evaluating the infestation of barley cultivars. Amounts of the pathogen DNA differed in six barley cultivars infected by F. culmorum. Transcription of HvUGT13248 was related to DON content in the samples. Low pathogenic infection and low DON content were accompanied by increased Fusarium Tri10 transcription in resistant cv. Amulet. This finding confirmed our recent results and makes us propose using this change as a possible marker of barley resistance against Fusarium.

Abstract Background The wild relatives of crop species represent a potentially valuable source of novel genetic variation, particularly in the context of improving the crop’s level of tolerance to abiotic stress. The mechanistic basis of these tolerances remains largely unexplored. Here, the focus was to characterize the transcriptomic response of the nodes (meristematic tissue) of couch grass (a relative of barley) to dehydration stress, and to compare it to that of the barley crown formed by both a drought tolerant and a drought sensitive barley cultivar. Results Many of the genes up-regulated in the nodes by the stress were homologs of genes known to be mediated by abscisic acid during the response to drought, or were linked to either development or lipid metabolism. Transporters also featured prominently, as did genes acting on root architecture. The resilience of the couch grass node arise from both their capacity to develop an altered, more effective root architecture, but also from their formation of a lipid barrier on their outer surface and their ability to modify both their lipid metabolism and transporter activity when challenged by dehydration stress. Conclusions Our analysis revealed the nature of dehydration stress response in couch grass. We suggested the tolerance is associated with lipid metabolism, the induction of transporters and the re-programming of development coordinated by ABA. We also proved the applicability of barley microarray for couch grass stress-response analysis.

DNA extraction is a crucial step in PCR analysis especially when analysing food samples that can be degraded and can potentially contain PCR-inhibiting substances. In this study, we compared the suitability of three DNA extraction methods - two kits: DNeasy® Plant Mini Kit and NucleoSpin® Food, and the CTAB method - for DNA extraction from commercial fruit jams. Fourteen jams with different contents of fruit, sugar and other additives were extracted in triplicate using the above-mentioned methods directly and after a washing step. The concentration and optical density were analysed using UV spectrophotometry and the amplifiability of the obtained DNA was evaluated using a PCR assay targeting a sequence coding for chloroplast tRNA-Leu. Samples isolated using the NucleoSpin® Food kit contained non-amplifiable DNA in eight cases, and samples isolated using the CTAB method could not be quantified. The DNeasy® Plant Mini Kit thus proved to be the most suitable method, since well-amplifiable DNA was obtained for all the analysed samples.

We present a successful use of the plasmid inhibition detection and DNA isolation protocol optimisation for four food/feed samples in qPCR analysis of the sequence coding for chloroplast tRNA-Leu: two meat meal samples and two samples made of cranberries (jam and dried fruit). The quantitative real-time polymerase chain reaction (qPCR) can be inhibited by various substances and the DNA content in the sample can be underestimated. It is necessary to identify the PCR inhibition and choose an optimal DNA isolation protocol to correctly evaluate the sample. In a previous study, we have developed an assay using plasmid DNA carrying a non-homologous random sequence identifying possible inhibitors in qPCR in food/feed samples. The plasmid assay allowed to effectively reveal the PCR inhibition in all of the different sample matrices and to choose an optimal DNA isolation protocol.

Six onion cultivars Bingo, Dormo, Elenka, Elbrus, Spirit, and Sturon grown in the Czech Republic for commercial purposes were analysed to investigate the content of health-promoting compounds. The results showed that at harvest time, cysteine sulfoxide content varied from 32.38 to 44.16 g/kg of dry weight, polyphenol content was between 2.66 and 3.37 g/kg of dry weight, and antioxidant activity ranged from 0.75 to 0.83 g/kg. Cv. Bingo had the highest level of the analysed compounds. The cultivars were concurrently analysed by DNA (microsatellite) markers. Dendrograms based on the chemical composition and DNA analysis were almost identical. This finding confirms the dependence of the secondary metabolite content on onion genotype.

Malting barley grains are essential components in the beer production. Fusarium infection can have severe effects on malt and beer, because it may inhibit the enzymatic activity in malt and may induce the occurrence of gushing and changes in the colour and flavour of the finished beer. We examined the growth of the filamentous fungi Fusarium culmorum in artificially infected and non-infected barley malting grains during the first steps of the malting process and under the effects of fungicide pretreatment (Hutton and Prosaro 250 EC) of barley plants. Our study focused on the fungi growth in two distinct barley malting cultivars Bojos and Malz. Fusarium growth was investigated by quantitative real-time PCR using TagMan MGB probes. Furthermore, we focused on the Tri5 and Tri6 genes because they play the most important roles in trichothecene biosynthesis. Surprisingly, the higher transcription activity of the Tri genes was found in the fungicide-treated cultivar Malz as compared with untreated cultivars.

The onion (Allium cepa L.) constitutes an important part of the human diet and is of economic importance on the vegetable market. Methods that allow unambiguous differentiation of cultivars and breeding lines are required for market control and the protection of plant breeders' rights. DNA analyses, such as single sequence repeat polymorphism (SSRs), represent such a tool. Based on the evaluation of polymorphism in 21 SSR loci, we established a panel of 15 easy to score SSR markers that differentiated 16 commercial onion cultivars in the Czech Republic. The polymorphism ranged from 2-3 alleles per locus, indicating a low level of diversity among the cultivars. The panel can also be used to evaluate genetic resources.

Garlic, Allium sativum L., is a vegetable long used for culinary and medical purposes. A certain level of garlic quality is required by the local consumers, which is usually preserved by the varieties grown in that region. The aim was to establish an assay offering fast and inexpensive differentiation of garlic varieties. Length polymorphism of microsatellite loci (SSR, ILP markers) is often used in such a case. No assays have been described earlier. A set of SSR and newly used ILP markers has been assembled and verified. SSR loci ASM53, ASM072, ASA08 and ASA17 were the most polymorphic. Up to 18 alleles were scored per these loci. Monomorphic loci were identified, and excluded from the assay. The assay allows for the authenticity and confirmation of Czech garlic varieties. Moreover, a cluster analysis separated the Czech bolting varieties, indicating their specific genetic basis. The breeding potential of contemporary garlic varieties and lines is discussed.

The feasibility of metabolomic fingerprinting approach based on ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UHPLC-QTOFMS) was studied to assess its ability to discriminate between maize varieties, and to show the associations between them on the metabolomic level. The non-targeted metabolomic analysis was applied to assess the variability within two varieties grown under different environmental conditions and to characterise the association within a sample set comprising both conventional and transgenic (MON-ØØ81Ø-6) maize varieties cultivated under the same environmental conditions (locality). Typical metabolomic fingerprints were established for individual plants. The plants representing two varieties formed well separated clusters. Metabolomic fingerprints of the second sample set enabled their unambiguous discrimination. The differences in metabolomic fingerprints between maize varieties were identified and documented by grouping in PCA and/or CA. The results indicate a similar genetic basis of transgenic maize varieties as they descend from a MON 810 event. The results explicitly showed that the variability of the metabolites in MON 810 did not exceed the ranges measured within the conventional varieties, thus supporting the concept of substantial equivalence.

Winter barley is an economically important cereal crop grown in higher latitudes and altitudes where low temperatures represent an important environmental constraint limiting crop productivity. In this study changes in proteome of leaves and crowns in a frost tolerant winter barley cv. Luxor in relation to short and long term periods of cold followed by a brief frost treatment were studied in order to disclose proteins responsible for the cold hardening process in distinct plant tissues. The mentioned changes have been monitored using two dimensional difference gel electrophoresis (2D-DIGE) with subsequent peptide-mapping protein identification. Regarding approximately 600–700 distinct protein spots detected on 2D gels, there has been found at least a two-fold change after exposure to low temperatures in about 10% of proteins in leaves and 13% of proteins in crowns. Protein and nitrogen metabolic processes have been influenced by low temperature to a similar extent in both tissues while catabolism, carbohydrate metabolism and proteins involved in stress response have been more affected in crowns than in leaves. The range of changes in protein abundance was generally higher in leaves and chloroplast proteins were frequently affected which suggests a priority to protect photosynthetic apparatus. Overall, our data proved existence of slightly different response strategies to low temperature stress in crowns and leaves, i.e., tissues with different biological role. Moreover, there have been found several proteins with large increase in accumulation, e.g., 33 kDa oxygen evolving protein of photosystem II in leaves and “enhanced disease susceptibility 1” in crowns; these proteins might have potential to indicate an enhanced level of frost tolerance in barley.

Genetically modified organisms (GMOs) entering the food chain have became its part, which is necessary to monitor. GMO analyses are used as a control mechanism according to valid acquis communautaire for traceability and labeling of GMOs. Generally, approved PCR based protocols are used and they require stepwise procedures that use amplification of species specific gene as initial point. This study aims to develop and verify PCR based assay for amplification of garden pea lectin gene (Pisum sativum L.) as reference one. Lectin gene was analysed in silico, selected region was amplified and sequenced and new set of species specific primers for identification of garden pea was designed. Conditions of conventional PCR as well as real-time PCR were optimised and specificity of new primer set on DNA extracted from garden pea cultivars as well as DNA extracted from other selected species from Fabaceae family was tested. Quantification of garden pea lectin gene using real-time PCR based on SYBR Green I was optimised and performance characteristics recorded. The characteristics fit to method acceptance criteria range. Plasmid with garden pea lectin sequence was developed and plasmid is available as a positive control.

The contents of three S-alk(en)ylcysteine sulfoxides (alliin, methiin, and isoalliin) were determined in the leaves, pseudostems, and bulbs of six garlic genotypes (two flowering plant morphotypes, two semi bolters, and two scape absent morphotypes) cultivated for five consecutive years at the same location. The average levels of alliin, methiin, and isoalliin were found to be as follows: 1.92, 0.44, and 0.07 mg/g fw in the leaves, 1.57, 0.27, and 0.08 mg/g fw in the pseudostems, and 1.71, 0.20 and 0.13 mg/g fw in the bulbs, respectively. No statistically significant year-to-year differences were observed between the samples. Furthermore, the total contents and relative proportions of S-alk(en)ylcysteine sulfoxides in various parts of the plants (leaves, pseudostems, bulbs and roots) were evaluated in detail during the whole vegetation period. It was observed that the total content of these amino acids gradually decreased in all parts except for the bulbs. In the bulbs, the total content initially decreased after planting but significantly increased in June and culminated before harvest. Analogous trends were also observed for alliin and methiin concentrations. On the other hand, isoalliin levels steadily decreased during the whole vegetation period in all parts of the plants.

The contents of major S-alk(en)ylcysteine sulfoxides (namely alliin, methiin and isoalliin) were determined in a set of 58 various garlic genotypes (22 flowering plant morphotypes, 14 semi bolting plants and 22 scape absent morphotype plants), representing the garlic collection of the Allium gene bank in the Czech Republic. The plants were cultivated in four successive years (2005-2008) and analysed immediately after harvest and subsequently after eight weeks of storage at 5°C. The total content of the three cysteine derivatives in fresh samples varied considerably between 3.35 mg/g fresh weigh and 12.77 mg/g fresh weight, with the mean of 7.50 mg/g fresh weight and the average relative proportions of alliin/methiin/isoalliin of 83/16/1. Upon 8-week storage at 5°C, the average total amount of S-alk(en)ylcysteine sulfoxides increased by 30% to 9.75 mg/g fresh weight, with the alliin/methiin/isoalliin ratio changing to 82/14/4. The data obtained were statistically evaluated using linear discrimination analysis to distinguish the differences between the years of harvest, between freshly harvested and stored samples, and between the individual morphotypes. While the year-to-year differences between the samples were statistically not very significant, the fresh and stored samples as well as the individual garlic morphotypes differed considerably in S-alk(en)ylcysteine sulfoxide content. Our results indicate that the content of S-alk(en)ylcysteine sulfoxides primarily depends on various genetic factors and post-harvest storage conditions, whereas the climatic conditions during the growth (e.g. temperature, irrigation) influence their level to a lesser extent. Various implications for the food and pharmaceutical industries are discussed.

Handling with genetically modified organisms (GMOs) is regulated namely in EC. Laboratories often use polymerase chain reaction (PCR) based screening methods to monitor the presence of GM particles in food commodities as a cost effective approach. The reliability was tested of such screening using 35S CaMV promoter as the target sequences. Soya grown from non-GM cultivar as declared by a seed company was investigated after the harvest, transport to the silo, and before processing. The results based on PCR and real-time PCR analysis clearly showed that, the contamination with debris of other species, dust during transport, storage, and other kind of handling led to contamination with detectable amounts of Cauliflower mosaic virus (CaMV). Impurities are allowed by EC regulations but may, as we have shown, interfere with the analytical procedures based on PCR. The identification of 35S CaMV promoter and NOS terminator in food with uncertain history and no approved specific events may indicate unknown GMOs and perhaps emergency situation.

The genetic diversity of 35 yellow-seeded Brassica napus L. accessions originating from China, Czech Republic and Poland was assessed by means of Amplified Fragment Length Polymorphism (AFLP) markers based on multiplex PCR using multi-colour fluorescent-labelled primers. Five brown-seeded accessions originating from China and France were selected as outliers. In total, 632 peaks were generated by AFLP reaction using 18 primer combinations. Only distinctly polymorphic markers among them were scored. In total, 242 polymorphic markers were detected with an average of 13.4 markers per primer combination. The AFLP analysis separated forty studied accessions into Chinese and European groups by UPGMA clustering and Principal Coordinates Analysis (PCA). The grouping of accessions based on the cluster analysis and PCA was generally consistent with known pedigree information and geographic origin. Notable geographical divergence was found between Chinese and European yellow-seeded accessions. This information is useful for yellow-seeded hybrid breeding and encouraging breeders to exchange their germplasm as to enlarge the genetic diversity of breeding accessions.

Reactions to artificial infection with Fusarium culmorum and (metconazole- or tebuconazole-based) fungicides were studied in nine winter wheat cultivars that were evaluated in field experiments at the location Prague-Ruzyne for four years (2001-2004) for deoxynivalenol (DON) content in grain, pathogen DNA content (Ct) by real-time quantitative PCR, percentage of Fusarium damaged grains (FDG), symptom scores and reductions in grain yield components. All examined traits were highly affected by conditions of experimental years and interactions with cultivars and treatments. Moderately resistant cultivars Arina and Petrus were included in the first homogeneous group in all traits, including the pathogen DNA content. To predict cultivar resistance to Fusarium head blight and accumulation of DON, the examination of the percentage of FDG in different environments appeared to be useful from practical aspects. The pathogen DNA content was significantly related to the content of DON under different conditions, however, the correlation coefficients ranged between 0.42 and 0.92. Different levels of DON could be detected at similar pathogen contents. The higher colonization of grain by the fungus was mostly connected with a strongly reduced amount of DON per pathogen unit (DON/Ct ratio). The fungicide treatment had a significant effect on a reduction in all traits except DON/Ct, but the effects on different traits were not often proportional and they were highly variable in the particular years (range 10-69%) and cultivars (range < 0-60%). While the application of fungicide caused a reduction in DON content in all cultivars, an increase in pathogen content after the application of fungicides was not exceptional. The low fungicide effect on a reduction in pathogen content was connected with higher temperatures (temperature extremes) in a 30-day period of disease development. The efficacy of fungicide treatment for DON was low at high pathogen content and late heading. The use of the collected data to improve control measures is discussed.

Reverse transcription coupled with real-time quantitative PCR (RT-qPCR) is a frequently used method for gene expression profiling. Reference genes (RGs) are commonly employed to normalize gene expression data. A limited information exist on the gene expression and profiling in developing barley caryopsis. Expression stability was assessed by measuring the cycle threshold (Ct) range and applying both the GeNorm (pair-wise comparison of geometric means) and Normfinder (model-based approach) principles for the calculation. Here, we have identified a set of four RGs suitable for studying gene expression in the developing barley caryopsis. These encode the proteins GAPDH, HSP90, HSP70 and ubiquitin. We found a correlation between the frequency of occurrence of a transcript in silico and its suitability as an RG. This set of RGs was tested by comparing the normalized level of β-amylase (β-amy1) transcript with directly measured quantities of the BMY1 gene product in the developing barley caryopsis. This panel of genes could be used for other gene expression studies, as well as to optimize β-amy1 analysis for study of the impact of β-amy1 expression upon barley end-use quality.

The fungi of the genus Fusarium occur worldwide mainly on cereal grains. They are considered to be not only plant-pathogenic, but also the producers of secondary metabolites - mycotoxins. In our study, we focused on the analysis by Q-PCR of two genes expression - Tri4, Tri10 - involved in the production of Fusarium mycotoxin deoxynivalenol. Reference genes β-tub and UBC were found to be suitable for the expression level normalisation using the program geNorm. The level of expression of the target genes was analysed on four isolates of Fusarium graminearum grown in vitro. In all four isolates, Tri10 gene expression level was lower than that of Tri4 gene expression.

Fusarium is a serious phytopathogenic fungal genus with producting of many kinds of highly toxic secondary metabolites - mycotoxins. The consumption of Fusarium contaminated food and feed can cause dangerous mycotoxicoses both in humans and animals, therefore the detection of a wider range of Fusarium species in the samples of crops is very important. The aim of our work was to test the reliability of detection and identification of three Fusarium species in infected wheat grains by DNA microarrays versus classical mycological methods and by specific PCR. The in-house DNA microarrays for the detection and identification of the selected Fusarium species by using oligonucleotides probes were prepared. For hybridisation on DNA microarrays fluorescent labelled PCR products were used of part of the translation elongation factor 1 alpha. The conditions of hybridisation were optimised on fungal template DNA. The method of DNA microarrays was verified on artificially infected samples of wheat and tested on unknown infected wheat samples with simultaneous analysis by classical mycological methods and by specific PCR.

Importance of the Polymerase chain reaction (PCR) have already crossed the border of mere target DNA sequence present or absence analysis. For number analyses e.g. Genetically Modified Organisms (GMOs) or gene expression assesment the DNA quantification is demanded. Real-time (or quantitative) PCR is the most used tool for nucleic acids quantification. PCR efficiency has relevant importance on DNA quantification - it should be almost same for each PCR and its value should varied between 90-100%. There are a lot of PCR enhancers and inhibitors well known. We described impact of used DNA solvent and used laboratory plastic on real-time PCR efficiency.