Fast and User-Friendly Detection of Flatfish Species (Pleuronectes platessa and Solea solea) via Loop-Mediated Isothermal Amplification (LAMP).

The detection of a Cytochrome b gene (cytb) for species differentiation in fish is intensively used. A fast alternative to expensive and time-consuming DNA barcoding is loop-mediated isothermal amplification (LAMP) in combination with efficient readout systems. For this reason, we developed LAMP assays for rapid species detection of Pleuronectes platessa and Solea solea, two economically important flatfish species in Europe that are prone to mislabeling. Species-specific primer sets targeting cytb were designed, and LAMP assays were optimized. With the optimized LAMP assays, we were able to detect up to 0.1 and 0.01 ng of target DNA of P. platessa and S. solea, respectively, and in each case up to 1% (w/w) of target species in mixtures with nontarget species. For future on-site detection, a lateral flow assay and a pocket-sized lab-on-phone assay were used as readout systems. The lab-on-phone assay with the S. solea specific primer set revealed cross-reactivity to Solea senegalensis. The assay targeting P. platessa proved to be highly specific. Both assays could be performed within 45 min and provided rapid and easy detection of fish species.

Identification of Cross-Species Marker Peptides for the Detection of Mammalian and Poultry Meat in Vegan and Vegetarian Foods.

Authentication of vegan and vegetarian foods is important since these increasingly popular food items could be adulterated with cheap meat to increase profit margins. In this study, nine marker peptides for the detection of meat (several species) were identified applying liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). These marker peptides enable the crucial differentiation of beef versus milk and chicken meat versus egg, demonstrated by the investigation of 19 commercial vegetarian meat substitutes containing milk and egg. Extensive experimental testing proved the presence of the cross-species meat marker peptides in 19 food-relevant types of mammals and poultry as well as their absence in more than 136 plant-based ingredients for the production of vegan and vegetarian foods. An authentic vegan sausage matrix based on an actual retail product was produced and spiked with 5.0%, w/w meat to confirm the high signal intensities and the heat stability of the marker peptides.

Essential terminology and considerations for validation of non-targeted methods.

Through their suggestive name, non-targeted methods (NTMs) do not aim at a predefined "needle in the haystack." Instead, they exploit all the constituents of the haystack. This new type of analytical method is increasingly finding applications in food and feed testing. However, the concepts, terms, and considerations related to this burgeoning field of analytical testing need to be propagated for the benefit of those associated with academic research, commercial development, or official control. This paper addresses frequently asked questions regarding terminology in connection with NTMs. The widespread development and adoption of these methods also necessitate the need to develop innovative approaches for NTM validation, i.e., evaluating the performance characteristics of a method to determine if it is fit-for-purpose. This work aims to provide a roadmap for approaching NTM validation. In doing so, the paper deliberates on the different considerations that influence the approach to validation and provides suggestions therefor.

Comparison of two commercial methods for smooth-shelled mussels (Mytilus spp.) species identification.

Seafood international trade has increased the labeling requirements in standards and regulations to include product information that enable traders and consumers to make informed choices. The European Union (EU) Regulation No. 1379/2013 imposes the declaration of an official commercial designation and scientific names for all the fishery and aquaculture products to be offered for sale to the final consumers. DNA analyses are used to enforce this regulation and to test authenticity in processed foods. We compared the performance of two mono-locus approaches for species identification (SI) in 61 Mytilus mussels: the high-resolution melting analysis of the polyphenolic adhesive protein gene and the partial sequencing of the histone H1C gene. The H1C sequences were analyzed with five different methods. Both approaches show discrepancies in the identification of putative hybrids (0.0 < κ < 0.687 and 0.0 < MCC < 0.724). Excluding putative hybrids, methods show substantial to perfect agreement (0.772 < κ < 1.0 and 0.783 < MCC < 1.0). This study highlights the need to use standardized molecular tools, as well as to use multi-locus methods for SI of Mytilus mussels in testing laboratories.

Structure of GTP cyclohydrolase I from Listeria monocytogenes, a potential anti-infective drug target.

A putative open reading frame encoding GTP cyclohydrolase I from Listeria monocytogenes was expressed in a recombinant Escherichia coli strain. The recombinant protein was purified and was confirmed to convert GTP to dihydroneopterin triphosphate (Km = 53 µM; vmax = 180 nmol mg-1 min-1). The protein was crystallized from 1.3 M sodium citrate pH 7.3 and the crystal structure was solved at a resolution of 2.4 Š(Rfree = 0.226) by molecular replacement using human GTP cyclohydrolase I as a template. The protein is a D5-symmetric decamer with ten topologically equivalent active sites. Screening a small library of about 9000 compounds afforded several inhibitors with IC50 values in the low-micromolar range. Several inhibitors had significant selectivity with regard to human GTP cyclohydrolase I. Hence, GTP cyclohydrolase I may be a potential target for novel drugs directed at microbial infections, including listeriosis, a rare disease with high mortality.

Species identification in mixed tuna samples with next-generation sequencing targeting two short cytochrome b gene fragments.

Conventional Sanger sequencing of PCR products is the gold standard for species authentication of seafood products. However, this method is inappropriate for the analysis of products that might contain mixtures of species, such as tinned tuna. The purpose of this study was to test whether next-generation sequencing (NGS) can be a solution for the authentication of mixed products. Nine tuna samples containing mixtures of up to four species were prepared and subjected to an NGS approach targeting two short cytochrome b gene (cytb) fragments on the Illumina MiSeq platform. Sequence recovery was precise and admixtures of as low as 1% could be identified, depending on the species composition of the mixtures. Duplicate samples as well as two individual NGS runs produced very similar results. A first test of three commercial tinned tuna samples indicated the presence of different species in the same tin, although this is forbidden by EU law.

Development of a Multiplex Real-Time PCR for Determination of Apricot in Marzipan Using the Plexor System.

Marzipan is a confectionary which is mostly offered in form of filled chocolate, pralines, or pure. According to the German guidelines for oil seeds only almonds, sugar and water are admitted ingredients of marzipan. A product very similar in taste is persipan which is used in the confectionary industry because of its stronger flavor. For persipan production almonds are replaced by debittered apricot or peach kernels. To guarantee high quality products for consumers, German raw paste producers have agreed a limit of apricot kernels in marzipan raw paste of 0.5%. Different DNA-based methods for quantitation of persipan contaminations in marzipan are already published. To increase the detection specificity compared to published intercalation dye-based assays, the present work demonstrate the utilization of a multiplex real-time PCR based on the Plexor technology. Thus, the present work enables the detection of at least 0.1% apricot DNA in almond DNA or less. By analyzing DNA mixtures, the theoretical limit of quantification of the duplex PCR for the quantitation of persipan raw paste DNA in marzipan raw paste DNA was determined as 0.05%.

Food Sensing: Aptamer-Based Trapping of Bacillus cereus Spores with Specific Detection via Real Time PCR in Milk.

Aerobic spores pose serious problems for both food product manufacturers and consumers. Milk is particularly at risk and thus an important issue of preventive consumer protection and quality assurance. The spore-former Bacillus cereus is a food poisoning Gram-positive pathogen which mainly produces two different types of toxins, the diarrhea inducing and the emetic toxins. Reliable and rapid analytical assays for the detection of B. cereus spores are required, which could be achieved by combining in vitro generated aptamers with highly specific molecular biological techniques. For the development of routine bioanalytical approaches, already existing aptamers with high affinity to B. cereus spores have been characterized by surface plasmon resonance (SPR) spectroscopy and fluorescence microscopy in terms of their dissociation constants and selectivity. Dissociation constants in the low nanomolar range (from 5.2 to 52.4 nM) were determined. Subsequently, the characterized aptamers were utilized for the establishment and validation of an aptamer-based trapping technique in both milk simulating buffer and milk with fat contents between 0.3 and 3.5%. Thereby, enrichment factors of up to 6-fold could be achieved. It could be observed that trapping protocol and characterized aptamers were fully adaptable to the application in milk. Due to the fact that aptamer selectivity is limited, a highly specific real time PCR assay was utilized following trapping to gain a higher degree of selectivity.

Catalysis of an Essential Step in Vitamin†B2 Biosynthesis by a Consortium of Broad Spectrum Hydrolases.

An enzyme catalysing the essential dephosphorylation of the riboflavin precursor, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate (6), was purified about 800-fold from a riboflavin-producing Bacillus subtilis strain, and was assigned as the translation product of the ycsE gene by mass spectrometry. YcsE is a member of the large haloacid dehalogenase (HAD) superfamily. The recombinant protein was expressed in Escherichia coli. It catalyses the hydrolysis of 6 (vmax , 12 µmol mg(-1) min(-1) ; KM , 54 µm) and of FMN (vmax , 25 µmol mg(-1) min(-1) ; KM , 135 µm). A ycsE deletion mutant of B. subtilis was not riboflavin dependent. Two additional proteins (YwtE, YitU) that catalyse the hydrolysis of 6 at appreciable rates were identified by screening 13 putative HAD superfamily members from B. subtilis. The evolutionary processes that have resulted in the handling of an essential step in the biosynthesis of an essential cofactor by a consortium of promiscuous enzymes require further analysis.

Food Fingerprinting: Characterization of the Ecuadorean Type CCN-51 of Theobroma cacao L. Using Microsatellite Markers.

The cocoa type "Colección Castro Naranjal 51" (CCN-51) is known for its resistance to specific climate conditions and its high yield, but it shows a weaker flavor profile and therefore is marketed as bulk cocoa. In a previous study, the two cocoa types Arriba and CCN-51 could easily be distinguished, but differences among the CCN-51 samples were observed. This was unexpected, as CCN-51 is reported to be a clone. To confirm whether CCN-51 is a pure clone, 10 simple sequence repeats (SSR) located on the nuclear genome were used to analyze various CCN-51 samples in comparison to the cocoa varieties Arriba and Criollo. As expected, there are differences in the SSR pattern among CCN-51, Arriba, and Criollo, but a variability within the CCN-51 sample set was detected as well. The previously described sequence variation in the chloroplast genome was confirmed by a variability in the microsatellite loci of the nuclear genome for a comprehensive cultivar collection of CCN-51 of both bean and leaf samples. In summary, beneath somaclonal variation, misidentification of plant collections and also sexual reproduction of CCN-51 can be suggested.

Food sensing: selection and characterization of DNA aptamers to Alicyclobacillus spores for trapping and detection from orange juice.

The quality of the beverage industry's products has to be constantly monitored to fulfill consumers' high expectations. The thermo-acidophilic Gram-positive Alicyclobacillus spp. are not pathogenic, but their heat-resistant endospores can survive juice-processing conditions and have become a major economic concern for the fruit juice industry. Current detection methods rely on cultivation, isolation, and organism identification, which can take up to a week, resulting in economic loss. This work presents the selection and identification of DNA aptamers targeting Alicyclobacillus spores by spore-SELEX (systematic evolution of ligands by exponential enrichment) in orange-juice-simulating buffer. The selection process was verified by various techniques, including flow cytometric binding assays, radioactive binding assays, and agarose gel electrophoresis. The subsequent aptamer characterization included the determination of dissociations constants and selectivity by different techniques, such as surface plasmon resonance spectroscopy and fluorescence microscopy. In summary, 10 different aptamers with an affinity to Alicyclobacillus spp. have been developed, analyzed, and characterized in terms of affinity and specificity.

DNA-based differentiation of the Ecuadorian cocoa types CCN-51 and Arriba based on sequence differences in the chloroplast genome.

Two cocoa types, Arriba and CCN-51, are being cultivated in Ecuador. With regard to the unique aroma, Arriba is considered a fine cocoa type, while CCN-51 is a bulk cocoa because of its weaker aroma. Because it is being assumed that Arriba is mixed with CCN-51, there is an interest in the analytical differentiation of the two types. Two methods to identify CCN-51 adulterations in Arriba cocoa were developed on the basis of differences in the chloroplast DNA. On the one hand, a different repeat of the sequence TAAAG in the inverted repeat region results in a different length of amplicons for the two cocoa types, which can be detected by agarose gel electrophoresis, capillary gel electrophoresis, and denaturing high-performance liquid chromatography. On the other hand, single nucleotide polymorphisms (SNPs) between the CCN-51 and Arriba sequences represent restriction sites, which can be used for restriction fragment length polymorphism analysis. A semi-quantitative analysis based on these SNPs is feasible. A method for an exact quantitation based on these results is not realizable. These sequence variations were confirmed for a comprehensive cultivar collection of Arriba and CCN-51, for both bean and leaf samples.

Recent advances in riboflavin biosynthesis.

Riboflavin is biosynthesized from GTP and ribulose 5-phosphate. Whereas the early reactions conducing to 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate show significant taxonomic variation, the subsequent reaction steps are universal in all taxonomic kingdoms. With the exception of a hitherto elusive phosphatase, all enzymes of the pathway have been characterized in some detail at the structural and mechanistic level. Some of the pathway enzymes (GTP cycloyhdrolase II, 3,4-dihydroxy-2-butanone 4-phosphate synthase, riboflavin synthase) have exceptionally complex reaction mechanisms. The commercial production of the vitamin is now entirely based on highly productive fermentation processes. Due to their absence in animals, the pathway enzymes are potential targets for the development of novel anti-infective drugs.

Enzymes from the haloacid dehalogenase (HAD) superfamily catalyse the elusive dephosphorylation step of riboflavin biosynthesis.

The missing link: Studies on the biosynthesis of riboflavin have failed to characterise dephosphorylation of the intermediate 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5'-phosphate. We show that this reaction can be catalysed in Escherichia coli by YigB and YbjI and in plant chloroplasts by AtcpFHy1, which are members of the haloacid dehalogenase superfamily.

Real-time PCR assays for the quantitation of rDNA from apricot and other plant species in marzipan.

Marzipan or marzipan raw paste is a typical German sweet which is consumed directly or is used as an ingredient in the bakery industry/confectionery (e.g., in stollen) and as filling for chocolate candies. Almonds (blanched and pealed) and sugar are the only ingredients for marzipan production according to German food guidelines. Especially for the confectionery industry, the use of persipan, which contains apricot or peach kernels instead of almonds, is preferred due to its stronger aroma. In most of the companies, both raw pastes are produced, in most cases on the same production line, running the risk of an unintended cross contamination. Additionally, due to high almond market values, dilutions of marzipan with cheaper seeds may occur. Especially in the case of apricot and almond, the close relationship of both species is a challenge for the analysis. DNA based methods for the qualitative detection of apricot, peach, pea, bean, lupine, soy, cashew, pistachio, and chickpea in marzipan have recently been published. In this study, different quantitation strategies on the basis of real-time PCR have been evaluated and a relative quantitation method with a reference amplification product was shown to give the best results. As the real-time PCR is based on the high copy rDNA-cluster, even contaminations <1% can be reliably quantitated.

Loop-mediated isothermal amplification (LAMP): methods for plant species identification in food.

Loop-mediated isothermal amplification (LAMP) is a DNA-based analytical method that can be used as an isothermal alternative to polymerase chain reaction (PCR). In comparison to PCR, the advantage of LAMP is the possibility to perform the isothermal reaction without any sophisticated technical equipment; only a water bath is needed, and naked eye detection is sufficient. Up to now, an application of LAMP methods for the detection of even closely related plant species in food or feed matrices has not been described, whereas a large number of PCR methods for that topic are cited in the literature. The aim of the study was the evaluation of LAMP-based methods for plant species identification with respect to method parameters such as R(2), LOD, and LOQ. An existing (real-time) PCR method (for the detection of spices) was used for comparison. It could be shown that the developed LAMP methods have potential as alternative strategies to PCR in DNA-based analysis.

Loop-mediated isothermal amplification (LAMP)-based method for rapid mushroom species identification.

Toxic mushroom species, such as the death cap ( Amanita phalloides ), are responsible for most mushroom poisonings. In the present work, novel loop-mediated isothermal amplification (LAMP) assays were used for the differentiation of even closely related edible and toxic mushroom species. The applicability of these methods was tested by cross-reaction studies and analysis of spiked mushroom samples (raw and fried material). Contaminations at the level of 2% (w/w) could be detected in different mushroom blends. Three detection methods were used: agarose gel analysis, fluorimetric real-time detection, and visual detection by lateral flow dipsticks (LFD). The LAMP assay combined with LFD detection allows the identification of A. phalloides in about 2 h (including DNA extraction) at a very low level of technical equipment (micropestle, water bath, and mobile centrifuge), which makes this technique perfectly suited for on-site applications.

Use of polymorphisms in the γ-gliadin gene of spelt and wheat as a tool for authenticity control.

Partial sequencing of the γ-gliadin gene of 62 spelt and 14 soft wheat cultivars was performed. Fifty-six of the 62 spelt cultivars and 13 of the 14 soft wheat cultivars were shown to exhibit the typical spelt or soft wheat γ-gliadin sequence, respectively. Exceptions were ascribed to crossbreeding of soft wheat and spelt. Using the typical soft wheat γ-gliadin sequence, two alternative DNA-based analytical methods were developed for the detection and quantification of spelt flour "adulteration" with soft wheat. A simple and fast detection of soft wheat in spelt flours could be achieved by restriction fragment length (RFLP) analysis. In combination with lab-on-a-chip capillary gel electrophoresis (LOC-CE) the soft wheat proportion could be estimated. Heteroduplex formation served as additional confirmation for the presence of spelt besides soft wheat. Hence, RFLP-LOC-CE constitutes a perfect analysis tool for the quality control of cereal seeds and pure cultivars. A precise quantification of soft wheat "adulterations" in spelt flour down to 1% could be achieved by the developed real-time PCR method. The calibration parameters of the real-time PCR assay fulfilled the minimum performance requirements of the European Network of GMO (genetically modified organisms) Laboratories (ENGL).

Marzipan: polymerase chain reaction-driven methods for authenticity control.

According to German food guidelines, almonds are the only oilseed ingredient allowed for the production of marzipan. Persipan is a marzipan surrogate in which the almonds are replaced by apricot or peach kernels. Cross-contamination of marzipan products with persipan may occur if both products are produced using the same production line. Adulterations or dilutions, respectively, of marzipan with other plant-derived products, for example, lupine or pea, have also been found. Almond and apricot plants are closely related. Consequently, classical analytical methods for the identification/differentiation often fail or are not sensitive enough to quantify apricot concentrations below 1%. Polymerase chain reaction (PCR)-based methods have been shown to enable the differentiation of closely related plant species in the past. These methods are characterized by high specificity and low detection limits. Isolation methods were developed and evaluated especially with respect to the matrix marzipan in terms of yield, purity, integrity, and amplificability of the isolated DNA. For the reliable detection of apricot, peach, pea, bean, lupine, soy, cashew, pistachio, and chickpea, qualitative standard and duplex PCR methods were developed and established. The applicability of these methods was tested by cross-reaction studies and analysis of spiked raw pastes. Contaminations at the level of 0.1% could be detected.

DNA-based identification of spices: DNA isolation, whole genome amplification, and polymerase chain reaction.

Usually spices are identified morphologically using simple methods like magnifying glasses or microscopic instruments. On the other hand, molecular biological methods like the polymerase chain reaction (PCR) enable an accurate and specific detection also in complex matrices. Generally, the origins of spices are plants with diverse genetic backgrounds and relationships. The processing methods used for the production of spices are complex and individual. Consequently, the development of a reliable DNA-based method for spice analysis is a challenging intention. However, once established, this method will be easily adapted to less difficult food matrices. In the current study, several alternative methods for the isolation of DNA from spices have been developed and evaluated in detail with regard to (i) its purity (photometric), (ii) yield (fluorimetric methods), and (iii) its amplifiability (PCR). Whole genome amplification methods were used to preamplify isolates to improve the ratio between amplifiable DNA and inhibiting substances. Specific primer sets were designed, and the PCR conditions were optimized to detect 18 spices selectively. Assays of self-made spice mixtures were performed to proof the applicability of the developed methods.