Analytical and Bioanalytical Chemistry (v.396, #6)
GMO analysis – a complex and challenging undertaking by Hendrik Emons (1949-1950).
is Head of the Reference Materials Unit of the European Commissions’ Joint Research Centre, Institute for Reference Materials and Measurements (IRMM), located in Geel, Belgium. He is in charge of programmes on the development, production, and certification of reference materials for a very broad range of application areas from food and microbiological to environmental analysis and nuclear safeguards, from biotechnology (including GMOs) and clinical analysis to engineered materials and nanotechnology. He is, moreover, Associate Professor at the University of Duisburg-Essen, Germany, and is on various scientific advisory boards and committees, for instance acting as Chairman of the ISO Committee on Reference Materials (REMCO) and member of the Research Council of the European Metrology Research Programme (EMRP) and the Executive Committee of AOAC’s Technical Division on Reference Materials. He serves also on the Advisory Board of Analytical and Bioanalytical Chemistry.
Knowledge-technology-based discovery of unauthorized genetically modified organisms by Tom Ruttink; Dany Morisset; Bart Van Droogenbroeck; Nada Lavrač; Guy L. M. Van Den Eede; Jana Žel; Marc De Loose (1951-1959).
is a postdoctoral fellow in the Technology & Food Unit of the Institute for Agricultural and Fisheries Research (ILVO), Belgium. In recent years, his main research interest has focused on the development of novel analytical methods for the detection and identification of GMOs in the food and feed chain. He was involved in several national and European research projects on GMO detection technology. is a postdoctoral fellow in the Genetically Modified Organisms group at the National Institute of Biology, Slovenia. His main research interest is in the development of novel analytical methods for GMO detection and identification. He is involved in several national and European research projects on GMO detection technology. He is Head of the European project GMOseek: Development of screening methods for GMOs. He is also an active member of the European Network of GMO Laboratories is a postdoctoral fellow in the Technology & Food Unit of the Institute for Agricultural and Fisheries Research (ILVO), Belgium. He has been working for several years on the development of novel GM applications (e.g., third-generation crops—molecular farming) and follows existing biotechnology applications (first-generation crops—input characteristics, such as herbicide tolerance, and second-generation crops—output characteristics, e.g., Golden Rice). is Head of the Department of Knowledge Technologies at the Jožef Stefan Institute, and holds a part-time professorship at the University of Nova Gorica, Slovenia. Her main research interests are in machine learning, in particular relational data mining and applications in medicine, and in the management of networked organizations. She was Scientific Coordinator of the European Scientific Network in Inductive Logic Programming (ILPNET) and Co-coordinator of the Fifth Framework Programme project Data Mining and Decision Support for Business Competitiveness: A European Virtual Enterprise (SolEuNet). is Head of the Molecular Biology and Genomics Unit of the European Commission Joint Research Centre in Ispra, Italy. This unit was nominated Community Reference Laboratory for GM Food and Feed in 2003 with legal tasks including the validation of detection methods for GM labeling and traceability compliance, distribution of control samples, and provision of expert advice in cases of litigation. He is also Chairman of the European Network of GMO Laboratories, Chairman of the Scientific Organizing Committee of the Global Conferences on GMO Analysis, and a member of different advisory boards. is Head of the Genetically Modified Organisms group at the National Institute of Biology, Slovenia. She has been working in the field of plant biotechnology since the early days. In recent years, she has dedicated her work mostly to the establishment of an internationally harmonized system for GMO testing, as well as to further improvements and new testing approaches and methods. She is also an active member of the European Network of GMO Laboratories. is Scientific Director of the research group on product quality and innovation in the Technology and Food Unit of the Institute for Agricultural and Fisheries Research (ILVO), Belgium. He has been working for more than 10 years in the domain of GMO detection, characterization, identification, and quantification. He has been a member of the EFSA GMO Panel and is currently involved in discussions on the safety of GMOs within the Belgian Biosafety Council. He is a member of the European Network for GMO Laboratories and is a partner of the NRL for GMO testing in Belgium.
See what you eat—broad GMO screening with microarrays by Franz von Götz (1961-1967).
Despite the controversy of whether genetically modified organisms (GMOs) are beneficial or harmful for humans, animals, and/or ecosystems, the number of cultivated GMOs is increasing every year. Many countries and federations have implemented safety and surveillance systems for GMOs. Potent testing technologies need to be developed and implemented to monitor the increasing number of GMOs. First, these GMO tests need to be comprehensive, i.e., should detect all, or at least the most important, GMOs on the market. This type of GMO screening requires a high degree of parallel tests or multiplexing. To date, DNA microarrays have the highest number of multiplexing capabilities when nucleic acids are analyzed. This trend article focuses on the evolution of DNA microarrays for GMO testing. Over the last 7 years, combinations of multiplex PCR detection and microarray detection have been developed to qualitatively assess the presence of GMOs. One example is the commercially available DualChip® GMO (Eppendorf, Germany; http://www.eppendorf-biochip.com ), which is the only GMO screening system successfully validated in a multicenter study. With use of innovative amplification techniques, promising steps have recently been taken to make GMO detection with microarrays quantitative. Figure EU-validated GMO screening microarray
Keywords: Genetically modified organism; Microarray; Genetically modified organism screening; Multiplex; Food and feed testing; Validation
Towards future reference systems for GM analysis by Stefanie Trapmann; Philippe Corbisier; Heinz Schimmel; Hendrik Emons (1969-1975).
Despite the fact that the measurement unit for the quantification of GMOs in food and feed products has not yet been unambiguously agreed upon in Europe, international trade requires reliable GMO analysis measuring comparably the GMO content of products. The two reference systems, based either on mass fractions or on copy number ratios, and their metrological traceability chains are presented and discussed. It is concluded that, properly established and expressed, measurement results in copy number ratios can provide a metrologically sound reference system. In this case, certified reference materials used for calibration and quality control can be independent of each other and the uncertainty derived from calibration can correctly be included in the overall uncertainty of the GMO measurement. However, further efforts are required to establish this metrological system.
Keywords: PCR; GMO; Calibration; Traceability; Measurement systems; Copy number ratio; Mass fraction
Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits by Tigst Demeke; G. Ronald Jenkins (1977-1990).
Biotechnology-derived varieties of canola, cotton, corn and soybean are being grown in the USA, Canada and other predominantly grain exporting countries. Although the amount of farmland devoted to production of biotechnology-derived crops continues to increase, lingering concerns that unintended consequences may occur provide the EU and most grain-importing countries with justification to regulate these crops. Legislation in the EU requires traceability of grains/oilseeds, food and feed products, and labelling, when a threshold level of 0.9% w/w of genetically engineered trait is demonstrated to be present in an analytical sample. The GE content is routinely determined by quantitative PCR (qPCR) and plant genomic DNA provides the template for the initial steps in this process. A plethora of DNA extraction methods exist for qPCR applications. Implementing standardized methods for detection of genetically engineered traits is necessary to facilitate grain marketing. The International Organization for Standardization draft standard 21571 identifies detergent-based methods and commercially available kits that are widely used for DNA extraction, but also indicates that adaptations may be necessary depending upon the sample matrix. This review assesses advantages and disadvantages of various commercially available DNA extraction kits, as well as modifications to published cetyltrimethylammonium bromide methods. Inhibitors are a major obstacle for efficient amplification in qPCR. The types of PCR inhibitors and techniques to minimize inhibition are discussed. Finally, accurate quantification of DNA for applications in qPCR is not trivial. Many confounders contribute to differences in analytical measurements when a particular DNA quantification method is applied and different methods do not always provide concordant results on the same DNA sample. How these differences impact measurement uncertainty in qPCR is considered.
Keywords: DNA extraction; Sample matrices; DNA quantification; PCR inhibitors; Variability of DNA yield
New approaches in GMO detection by Maddalena Querci; Marc Van den Bulcke; Jana Žel; Guy Van den Eede; Hermann Broll (1991-2002).
The steady rate of development and diffusion of genetically modified plants and their increasing diversification of characteristics, genes and genetic control elements poses a challenge in analysis of genetically modified organisms (GMOs). It is expected that in the near future the picture will be even more complex. Traditional approaches, mostly based on the sequential detection of one target at a time, or on a limited multiplexing, allowing only a few targets to be analysed at once, no longer meet the testing requirements. Along with new analytical technologies, new approaches for the detection of GMOs authorized for commercial purposes in various countries have been developed that rely on (1) a smart and accurate strategy for target selection, (2) the use of high-throughput systems or platforms for the detection of multiple targets and (3) algorithms that allow the conversion of analytical results into an indication of the presence of individual GMOs potentially present in an unknown sample. This paper reviews the latest progress made in GMO analysis, taking examples from the most recently developed strategies and tools, and addresses some of the critical aspects related to these approaches.
Keywords: Genetically modified organism; Genetically modified organism detection; Detection strategies; High-throughput systems; Matrix approach; Decision support systems
Effect of food processing on plant DNA degradation and PCR-based GMO analysis: a review by Nicolas Gryson (2003-2022).
The applicability of a DNA-based method for GMO detection and quantification depends on the quality and quantity of the DNA. Important food-processing conditions, for example temperature and pH, may lead to degradation of the DNA, rendering PCR analysis impossible or GMO quantification unreliable. This review discusses the effect of several food processes on DNA degradation and subsequent GMO detection and quantification. The data show that, although many of these processes do indeed lead to the fragmentation of DNA, amplification of the DNA may still be possible. Length and composition of the amplicon may, however, affect the result, as also may the method of extraction used. Also, many techniques are used to describe the behaviour of DNA in food processing, which occasionally makes it difficult to compare research results. Further research should be aimed at defining ingredients in terms of their DNA quality and PCR amplification ability, and elaboration of matrix-specific certified reference materials.
Keywords: Food processing; DNA degradation; GMO detection; DNA quantification; PCR analysis
Comparison of nine different real-time PCR chemistries for qualitative and quantitative applications in GMO detection by Meti Buh Gašparič; Torstein Tengs; Jose Luis La Paz; Arne Holst-Jensen; Maria Pla; Teresa Esteve; Jana Žel; Kristina Gruden (2023-2029).
Several techniques have been developed for detection and quantification of genetically modified organisms, but quantitative real-time PCR is by far the most popular approach. Among the most commonly used real-time PCR chemistries are TaqMan probes and SYBR green, but many other detection chemistries have also been developed. Because their performance has never been compared systematically, here we present an extensive evaluation of some promising chemistries: sequence-unspecific DNA labeling dyes (SYBR green), primer-based technologies (AmpliFluor, Plexor, Lux primers), and techniques involving double-labeled probes, comprising hybridization (molecular beacon) and hydrolysis (TaqMan, CPT, LNA, and MGB) probes, based on recently published experimental data. For each of the detection chemistries assays were included targeting selected loci. Real-time PCR chemistries were subsequently compared for their efficiency in PCR amplification and limits of detection and quantification. The overall applicability of the chemistries was evaluated, adding practicability and cost issues to the performance characteristics. None of the chemistries seemed to be significantly better than any other, but certain features favor LNA and MGB technology as good alternatives to TaqMan in quantification assays. SYBR green and molecular beacon assays can perform equally well but may need more optimization prior to use.
Keywords: Real-time PCR; GMO detection; TaqMan; SYBR green; Molecular beacons; Alternative chemistries
Development of sampling approaches for the determination of the presence of genetically modified organisms at the field level by Jelka Šuštar-Vozlič; Katja Rostohar; Andrej Blejec; Petra Kozjak; Zoran Čergan; Vladimir Meglič (2031-2041).
In order to comply with the European Union regulatory threshold for the adventitious presence of genetically modified organisms (GMOs) in food and feed, it is important to trace GMOs from the field. Appropriate sampling methods are needed to accurately predict the presence of GMOs at the field level. A 2-year field experiment with two maize varieties differing in kernel colour was conducted in Slovenia. Based on the results of data mining analyses and modelling, it was concluded that spatial relations between the donor and receptor field were the most important factors influencing the distribution of outcrossing rate (OCR) in the field. The approach for estimation fitting function parameters in the receptor (non-GM) field at two distances from the donor (GM) field (10 and 25 m) for estimation of the OCR (GMO content) in the whole receptor field was developed. Different sampling schemes were tested; a systematic random scheme in rows was proposed to be applied for sampling at the two distances for the estimation of fitting function parameters for determination of OCR. The sampling approach had already been validated with some other OCR data and was practically applied in the 2009 harvest in Poland. The developed approach can be used for determination of the GMO presence at the field level and for making appropriate labelling decisions. The importance of this approach lies in its possibility to also address other threshold levels beside the currently prescribed labelling threshold of 0.9% for food and feed.
Keywords: Maize; GMO; Outcrossing rate; Sampling; Field; Labelling
Development of a qualitative, multiplex real-time PCR kit for screening of genetically modified organisms (GMOs) by Hans-Henno Dörries; Ivonne Remus; Astrid Grönewald; Cordt Grönewald; Kornelia Berghof-Jäger (2043-2054).
The number of commercially available genetically modified organisms (GMOs) and therefore the diversity of possible target sequences for molecular detection techniques are constantly increasing. As a result, GMO laboratories and the food production industry currently are forced to apply many different methods to reliably test raw material and complex processed food products. Screening methods have become more and more relevant to minimize the analytical effort and to make a preselection for further analysis (e.g., specific identification or quantification of the GMO). A multiplex real-time PCR kit was developed to detect the 35S promoter of the cauliflower mosaic virus, the terminator of the nopaline synthase gene of Agrobacterium tumefaciens, the 35S promoter from the figwort mosaic virus, and the bar gene of the soil bacterium Streptomyces hygroscopicus as the most widely used sequences in GMOs. The kit contains a second assay for the detection of plant-derived DNA to control the quality of the often processed and refined sample material. Additionally, the plant-specific assay comprises a homologous internal amplification control for inhibition control. The determined limits of detection for the five assays were 10 target copies/reaction. No amplification products were observed with DNAs of 26 bacterial species, 25 yeasts, 13 molds, and 41 not genetically modified plants. The specificity of the assays was further demonstrated to be 100% by the specific amplification of DNA derived from reference material from 22 genetically modified crops. The applicability of the kit in routine laboratory use was verified by testing of 50 spiked and unspiked food products. The herein described kit represents a simple and sensitive GMO screening method for the reliable detection of multiple GMO-specific target sequences in a multiplex real-time PCR reaction. Figure Real-time amplification curves for the event MON810 with 1,000, 100, 10, and 1 target copies/reaction of the 35S promoter of the cauliflower mosaic virus.
Keywords: Genetically modified organisms; GMO; Real-time PCR; Multiplex PCR; Screening
Establishment of a system based on universal multiplex-PCR for screening genetically modified crops by I-Jen Lu; Chih-Hui Lin; Tzu-Ming Pan (2055-2064).
The rapid development of many genetically modified (GM) crops in the past two decades makes it necessary to introduce an alternative strategy for routine screening and identification. In this study, we established a universal multiplex PCR detection system which will effectively reduce the number of reactions needed for sample identification. The PCR targets of this system include the six most frequently used transgenic elements: cauliflower mosaic virus (CaMV) 35S promoter, Agrobacterium tumefaciens nopaline synthase (nos) promoter, Agrobacterium tumefaciens nopaline synthase (nos) terminator, the neomycin phosphotransferase II (nptII) gene, the 5-enolpyruvylshikimate-3-phosphate synthase (CP4 epsps) gene of Agrobacterium tumefaciens strain CP4, and the phosphinothricin N-acetyltransferase (pat) gene. According to the AGBIOS database, the coverage of this detection system is 93% of commercial GM crops. This detection system could detect all certified reference materials (CRMs) at the 1.0% level. The correct combination of all the CRM amplicon patterns proved the specificity of this multiplex PCR system. Furthermore, the amplicon patterns of this multiplex PCR detection system could be used as an index of classification which will narrow the range of possible GM products. The simulation result of this multiplex PCR detection system on all commercialized 139 GM products in the AGBIOS database showed that the maximum number of PCR reactions needed to identify an unknown sample can be reduced to 13. In this study, we established a high-throughput multiplex PCR detection system with feasible sensitivity, specificity, and cost. By incorporating this detection system, the routine GM crop-detection process will meet the challenges resulting from a rapid increase in the number of GM crops in the future.
Keywords: GMO; Multiplex PCR; Detection system; Transgenic element; Universal screening system
A practical approach to screen for authorised and unauthorised genetically modified plants by Hans-Ulrich Waiblinger; Lutz Grohmann; Joachim Mankertz; Dirk Engelbert; Klaus Pietsch (2065-2072).
In routine analysis, screening methods based on real-time PCR are most commonly used for the detection of genetically modified (GM) plant material in food and feed. In this paper, it is shown that the combination of five DNA target sequences can be used as a universal screening approach for at least 81 GM plant events authorised or unauthorised for placing on the market and described in publicly available databases. Except for maize event LY038, soybean events DP-305423 and BPS-CV127-9 and cotton event 281-24-236 × 3006-210-23, at least one of the five genetic elements has been inserted in these GM plants and is targeted by this screening approach. For the detection of these sequences, fully validated real-time PCR methods have been selected. A screening table is presented that describes the presence or absence of the target sequences for most of the listed GM plants. These data have been verified either theoretically according to available databases or experimentally using available reference materials. The screening table will be updated regularly by a network of German enforcement laboratories.
Keywords: Genetically modified organism (GMO); Screening table; Real-time PCR; Unauthorised GMO; GMO matrix; Placing on the market; Genetically modified food; Genetically modified feed
Molecular toolbox for the identification of unknown genetically modified organisms by Tom Ruttink; Rolinde Demeyer; Elke Van Gulck; Bart Van Droogenbroeck; Maddalena Querci; Isabel Taverniers; Marc De Loose (2073-2089).
Competent laboratories monitor genetically modified organisms (GMOs) and products derived thereof in the food and feed chain in the framework of labeling and traceability legislation. In addition, screening is performed to detect the unauthorized presence of GMOs including asynchronously authorized GMOs or GMOs that are not officially registered for commercialization (unknown GMOs). Currently, unauthorized or unknown events are detected by screening blind samples for commonly used transgenic elements, such as p35S or t-nos. If (1) positive detection of such screening elements shows the presence of transgenic material and (2) all known GMOs are tested by event-specific methods but are not detected, then the presence of an unknown GMO is inferred. However, such evidence is indirect because it is based on negative observations and inconclusive because the procedure does not identify the causative event per se. In addition, detection of unknown events is hampered in products that also contain known authorized events. Here, we outline alternative approaches for analytical detection and GMO identification and develop new methods to complement the existing routine screening procedure. We developed a fluorescent anchor-polymerase chain reaction (PCR) method for the identification of the sequences flanking the p35S and t-nos screening elements. Thus, anchor-PCR fingerprinting allows the detection of unique discriminative signals per event. In addition, we established a collection of in silico calculated fingerprints of known events to support interpretation of experimentally generated anchor-PCR GM fingerprints of blind samples. Here, we first describe the molecular characterization of a novel GMO, which expresses recombinant human intrinsic factor in Arabidopsis thaliana. Next, we purposefully treated the novel GMO as a blind sample to simulate how the new methods lead to the molecular identification of a novel unknown event without prior knowledge of its transgene sequence. The results demonstrate that the new methods complement routine screening procedures by providing direct conclusive evidence and may also be useful to resolve masking of unknown events by known events. Figure Molecular toolbox for the identification of genetically modified organisms
Keywords: GMO screening analysis; GMO identification; Anchor-PCR; Fingerprinting; p35S; t-nos
Detection and identification of multiple genetically modified events using DNA insert fingerprinting by Philippe Raymond; Louis Gendron; Moustafa Khalf; Sylvianne Paul; Kim L. Dibley; Somanath Bhat; Vicki R. D. Xie; Lina Partis; Marie-Eve Moreau; Cheryl Dollard; Marie-José Coté; Serge Laberge; Kerry R. Emslie (2091-2102).
Current screening and event-specific polymerase chain reaction (PCR) assays for the detection and identification of genetically modified organisms (GMOs) in samples of unknown composition or for the detection of non-regulated GMOs have limitations, and alternative approaches are required. A transgenic DNA fingerprinting methodology using restriction enzyme digestion, adaptor ligation, and nested PCR was developed where individual GMOs are distinguished by the characteristic fingerprint pattern of the fragments generated. The inter-laboratory reproducibility of the amplified fragment sizes using different capillary electrophoresis platforms was compared, and reproducible patterns were obtained with an average difference in fragment size of 2.4 bp. DNA insert fingerprints for 12 different maize events, including two maize hybrids and one soy event, were generated that reflected the composition of the transgenic DNA constructs. Once produced, the fingerprint profiles were added to a database which can be readily exchanged and shared between laboratories. This approach should facilitate the process of GMO identification and characterization.
Keywords: Genetically modified organism detection; GMO; Anchored PCR; Genome walking; Fingerprint
Validation of a newly developed hexaplex real-time PCR assay for screening for presence of GMOs in food, feed and seed by C. Bahrdt; A. B. Krech; A. Wurz; D. Wulff (2103-2112).
For years, an increasing number and diversity of genetically modified plants has been grown on a commercial scale. The need for detection and identification of these genetically modified organisms (GMOs) calls for broad and at the same time flexible high throughput testing methods. Here we describe the development and validation of a hexaplex real-time polymerase chain reaction (PCR) screening assay covering more than 100 approved GMOs containing at least one of the GMO targets of the assay. The assay comprises detection systems for Cauliflower Mosaic Virus 35S promoter, Agrobacterium tumefaciens NOS terminator, Figwort Mosaic Virus 34S promoter and two construct-specific sequences present in novel genetically modified soybean and maize that lack common screening elements. Additionally a detection system for an internal positive control (IPC) indicating the presence or absence of PCR inhibiting substances was included. The six real-time PCR systems were allocated to five detection channels showing no significant crosstalk between the detection channels. As part of an extensive validation, a limit of detection (LODabs) ≤ ten target copies was proven in hexaplex format. A sensitivity ≤ ten target copies of each GMO detection system was still shown in highly asymmetric target situations in the presence of 1,000 copies of all other GMO targets of each detection channel. Furthermore, the applicability to a broad sample spectrum and reliable indication of inhibition by the IPC system was demonstrated. The presented hexaplex assay offers sensitive and reliable detection of GMOs in processed and unprocessed food, feed and seed samples with high efficiency. Figure PCR amplification curves of all PCR systems comprised in the hexaplex real-time PCR assay for screening for presence of GMOs in food, feed and seed with 50 target copies per reaction.
Keywords: GMO; Multiplex; Hexaplex; Real-time PCR; Screening; Validation
A theoretical introduction to “Combinatory SYBR®Green qPCR Screening”, a matrix-based approach for the detection of materials derived from genetically modified plants by Marc Van den Bulcke; Antoon Lievens; Elodie Barbau-Piednoir; Guillaume MbongoloMbella; Nancy Roosens; Myriam Sneyers; Amaya Leunda Casi (2113-2123).
The detection of genetically modified (GM) materials in food and feed products is a complex multi-step analytical process invoking screening, identification, and often quantification of the genetically modified organisms (GMO) present in a sample. “Combinatory qPCR SYBR®Green screening” (CoSYPS) is a matrix-based approach for determining the presence of GM plant materials in products. The CoSYPS decision-support system (DSS) interprets the analytical results of SYBR®GREEN qPCR analysis based on four values: the C t- and T m values and the LOD and LOQ for each method. A theoretical explanation of the different concepts applied in CoSYPS analysis is given (GMO Universe, “Prime number tracing”, matrix/combinatory approach) and documented using the RoundUp Ready soy GTS40-3-2 as an example. By applying a limited set of SYBR®GREEN qPCR methods and through application of a newly developed “prime number”-based algorithm, the nature of subsets of corresponding GMO in a sample can be determined. Together, these analyses provide guidance for semi-quantitative estimation of GMO presence in a food and feed product.
Keywords: GMO; Screening; Prime number; qPCR; SYBRGreen
Characterization of polyadenylated cryIA(b) transcripts in maize MON810 commercial varieties by José Luis La Paz; Carlos Vicient; Pere Puigdomènech; Maria Pla (2125-2133).
The Zea mays L. event MON810 is one of the major commercialized genetically modified crops. The inserted expression cassette has a 3′ truncation partially affecting the cryIA(b) coding sequence, resulting in the lack of the NOS terminator, with transcription of the transgene reported to read-through 3′-past the truncation site. Here, we demonstrate that the cryIA(b) transgene gives rise to a variety of polyadenylated transcripts of different sizes that extend to around 1 kbp downstream the truncation site. A Stop codon at position +7 downstream the truncation site indicates the production of a transgenic protein with two additional amino acids; which is compatible with the reported size of the CryIA(b) protein in MON810. There is no evidence of the existence of other translated products. Several main 3′ transcription termination regions were detected close to the truncation site and in the transgene 3′ flanking sequence. Next to these main termination sites, we identified some sequence motifs that could potentially act as 3′-end-processing elements and drive termination of the transgene transcripts. The MON810 transgene has been introduced into different commercial varieties through breeding programs. Here, we demonstrate that there are no significant differences among the levels of transgene mRNA accumulation, major transcript sizes and 3′ termini profiles comparing a number of MON810 commercial varieties grown under similar environmental conditions. Commercial varieties of this event appear to be stable in terms of transgene expression. Figure Similar cryIA(b) RACE 3’-PCR profiles were obtained with mRNA from six MON810 commercial varieties: superposition of electropherograms and schematic representation of the length of transgene mRNA molecules.
Keywords: GMO (genetically modified organism); MON810 maize; Transgene mRNA; 3′-end-processing site; Polyadenylated mRNA
A preamplification approach to GMO detection in processed foods by S. Del Gaudio; A. Cirillo; G. Di Bernardo; U. Galderisi; M. Cipollaro (2135-2142).
DNA is widely used as a target for GMO analysis because of its stability and high detectability. Real-time PCR is the method routinely used in most analytical laboratories due to its quantitative performance and great sensitivity. Accurate DNA detection and quantification is dependent on the specificity and sensitivity of the amplification protocol as well as on the quality and quantity of the DNA used in the PCR reaction. In order to enhance the sensitivity of real-time PCR and consequently expand the number of analyzable target genes, we applied a preamplification technique to processed foods where DNA can be present in low amounts and/or in degraded forms thereby affecting the reliability of qualitative and quantitative results. The preamplification procedure utilizes a pool of primers targeting genes of interest and is followed by real-time PCR reactions specific for each gene. An improvement of Ct values was found comparing preamplified vs. non-preamplified DNA. The strategy reported in the present study will be also applicable to other fields requiring quantitative DNA testing by real-time PCR.
Keywords: GMO detection; Real-time PCR; Preamplification
Absolute quantification of genetically modified MON810 maize (Zea mays L.) by digital polymerase chain reaction by Philippe Corbisier; Somanath Bhat; Lina Partis; Vicki Rui Dan Xie; Kerry R. Emslie (2143-2150).
Quantitative analysis of genetically modified (GM) foods requires estimation of the amount of the transgenic event relative to an endogenous gene. Regulatory authorities in the European Union (EU) have defined the labelling threshold for GM food on the copy number ratio between the transgenic event and an endogenous gene. Real-time polymerase chain reaction (PCR) is currently being used for quantification of GM organisms (GMOs). Limitations in real-time PCR applications to detect very low number of DNA targets has led to new developments such as the digital PCR (dPCR) which allows accurate measurement of DNA copies without the need for a reference calibrator. In this paper, the amount of maize MON810 and hmg copies present in a DNA extract from seed powders certified for their mass content and for their copy number ratio was measured by dPCR. The ratio of these absolute copy numbers determined by dPCR was found to be identical to the ratios measured by real-time quantitative PCR (qPCR) using a plasmid DNA calibrator. These results indicate that both methods could be applied to determine the copy number ratio in MON810. The reported values were in agreement with estimations from a model elaborated to convert mass fractions into copy number fractions in MON810 varieties. This model was challenged on two MON810 varieties used for the production of MON810 certified reference materials (CRMs) which differ in the parental origin of the introduced GM trait. We conclude that dPCR has a high metrological quality and can be used for certifying GM CRMs in terms of DNA copy number ratio.
Keywords: PCR; Maize; Zea mays ; Genetically modified organism; Biochips high-throughput screening; Nucleic acids (DNA|RNA)
Design of multiplex calibrant plasmids, their use in GMO detection and the limit of their applicability for quantitative purposes owing to competition effects by Frédéric Debode; Aline Marien; Eric Janssen; Gilbert Berben (2151-2164).
Five double-target multiplex plasmids to be used as calibrants for GMO quantification were constructed. They were composed of two modified targets associated in tandem in the same plasmid : (1) a part of the soybean lectin gene and (2) a part of the transgenic construction of the GTS40-3-2 event. Modifications were performed in such a way that each target could be amplified with the same primers as those for the original target from which they were derived but such that each was specifically detected with an appropriate probe. Sequence modifications were done to keep the parameters of the new target as similar as possible to those of its original sequence. The plasmids were designed to be used either in separate reactions or in multiplex reactions. Evidence is given that with each of the five different plasmids used in separate wells as a calibrant for a different copy number, a calibration curve can be built. When the targets were amplified together (in multiplex) and at different concentrations inside the same well, the calibration curves showed that there was a competition effect between the targets and this limits the range of copy numbers for calibration over a maximum of 2 orders of magnitude. Another possible application of multiplex plasmids is discussed.
Keywords: Genetically modified organism; Quantification; Plasmid; Competition; Multiplex; Internal control
Use of pJANUS™-02-001 as a calibrator plasmid for Roundup Ready soybean event GTS-40-3-2 detection: an interlaboratory trial assessment by A. Lievens; G. Bellocchi; D. De Bernardi; W. Moens; C. Savini; M. Mazzara; G. Van den Eede; M. Van den Bulcke (2165-2173).
Owing to the labelling requirements of food and feed products containing materials derived from genetically modified organisms, quantitative detection methods have to be developed for this purpose, including the necessary certified reference materials and calibrator standards. To date, for most genetically modified organisms authorized in the European Union, certified reference materials derived from seed powders are being developed. Here, an assessment has been made on the feasibility of using plasmid DNA as an alternative calibrator for the quantitative detection of genetically modified organisms. For this, a dual-target plasmid, designated as pJANUS™-02-001, comprising part of a junction region of genetically modified soybean event GTS-40-3-2 and the endogenous soybean-specific lectin gene was constructed. The dynamic range, efficiency and limit of detection for the soybean event GTS-40-3-2 real-time quantitative polymerase chain reaction (Q-PCR) system described by Terry et al. (J AOAC Int 85(4):938–944, 2002) were shown to be similar for in house produced homozygous genomic DNA from leaf tissue of soybean event GTS-40-3-2 and for plasmid pJANUS™-02-001 DNA backgrounds. The performance of this real-time Q-PCR system using both types of DNA templates as calibrator standards in quantitative DNA analysis was further assessed in an interlaboratory trial. Statistical analysis and fuzzy-logic-based interpretation were performed on critical method parameters (as defined by the European Network of GMO Laboratories and the Community Reference Laboratory for GM Food and Feed guidelines) and demonstrated that the plasmid pJANUS™-02-001 DNA represents a valuable alternative to genomic DNA as a calibrator for the quantification of soybean event GTS-40-3-2 in food and feed products.
Keywords: Fuzzy logic; Genetically modified organisms; pJANUS; Real-time quantitative polymerase chain reaction; Validation
The use of 35S and Tnos expression elements in the measurement of genetically engineered plant materials by Marcia J. Holden; Marci Levine; Tandace Scholdberg; Ross J. Haynes; G. Ronald Jenkins (2175-2187).
An online survey was conducted by the International Life Sciences Institute, Food Biotechnology Committee, on the use of qualitative and quantitative polymerase chain reaction (PCR) assays for cauliflower mosaic virus 35S promoter and Agrobacterium tumefaciens Tnos DNA sequence elements for the detection of genetically engineered (GE) crop plant material. Forty-four testing laboratories around the world completed the survey. The results showed the widespread use of such methods, the multiplicity of published and in-house methods, and the variety of reference materials and calibrants in use. There was an interest on the part of respondents in validated quantitative assays relevant to all GE events that contain these two genetic elements. Data are presented by testing two variations each of five published real-time quantitative PCR methods for 35S detection on eight maize reference materials. The results showed that two of the five methods were not suitable for all the eight reference materials, with poor linear regression parameters and multiple PCR amplification products for some of the reference materials. This study demonstrates that not all 35S methods produce satisfactory results, emphasizing the need for method validation.
Keywords: Nucleic acids (DNA); PCR; CaMV 35S promoter; Tnos ; Quantitative real-time PCR; Biotechnological products
Evaluation of the reliability of maize reference assays for GMO quantification by Nina Papazova; David Zhang; Kristina Gruden; Jana Vojvoda; Litao Yang; Meti Buh Gašparič; Andrej Blejec; Stephane Fouilloux; Marc De Loose; Isabel Taverniers (2189-2201).
A reliable PCR reference assay for relative genetically modified organism (GMO) quantification must be specific for the target taxon and amplify uniformly along the commercialised varieties within the considered taxon. Different reference assays for maize (Zea mays L.) are used in official methods for GMO quantification. In this study, we evaluated the reliability of eight existing maize reference assays, four of which are used in combination with an event-specific polymerase chain reaction (PCR) assay validated and published by the Community Reference Laboratory (CRL). We analysed the nucleotide sequence variation in the target genomic regions in a broad range of transgenic and conventional varieties and lines: MON 810 varieties cultivated in Spain and conventional varieties from various geographical origins and breeding history. In addition, the reliability of the assays was evaluated based on their PCR amplification performance. A single base pair substitution, corresponding to a single nucleotide polymorphism (SNP) reported in an earlier study, was observed in the forward primer of one of the studied alcohol dehydrogenase 1 (Adh1) (70) assays in a large number of varieties. The SNP presence is consistent with a poor PCR performance observed for this assay along the tested varieties. The obtained data show that the Adh1 (70) assay used in the official CRL NK603 assay is unreliable. Based on our results from both the nucleotide stability study and the PCR performance test, we can conclude that the Adh1 (136) reference assay (T25 and Bt11 assays) as well as the tested high mobility group protein gene assay, which also form parts of CRL methods for quantification, are highly reliable. Despite the observed uniformity in the nucleotide sequence of the invertase gene assay, the PCR performance test reveals that this target sequence might occur in more than one copy. Finally, although currently not forming a part of official quantification methods, zein and SSIIb assays are found to be highly reliable in terms of nucleotide stability and PCR performance and are proposed as good alternative targets for a reference assay for maize. Figure Comparison of performance of reference assays in worldwide collection of varieties. Each scatter plot was constructed by plotting the copy numbers determined with one reference assay (x-axis) against the copy numbers determined with another reference assay (y-axis) after the genewise normalisation of copy number data. The red line in each scatter plot denotes a perfect correlation (r = 1) of copy numbers between the two assays. The individual correlation coefficients between the assays are also shown in the squares diagonally opposite to individual plots
Keywords: GMO; Real-time PCR quantification; Endogenous target taxon; Reference assay; Reliability
Detection of Cry1Ab toxin in the leaves of MON 810 transgenic maize by András Székács; Éva Lauber; Eszter Takács; Béla Darvas (2203-2211).
The distribution of Cry1Ab toxin was detected in the leaves of genetically modified maize of genetic event MON 810 by enzyme-linked immunosorbent assay. Cry1Ab toxin contents in the leaves at reproductive (milk, R3) phenological stage were measured to be between 3,878 and 11,148 ng Cry1Ab toxin/g fresh weight. Toxin content was significantly lesser (significant difference (SD) = 1,823 ng Cry1Ab toxin/g fresh leaf weight, p < 0.01) in leaves at the lowest leaf level, than at higher leaf levels, probably due to partial leaf necrotisation. A substantial (up to 22%) plant-to-plant variation in Cry1Ab contents in leaves was observed. When studying toxin distribution within the cross and longitudinal sections of single leaves, lesser variability was detected diagonally, with approximately 20% higher toxin concentrations at or near the leaf vein. More significant variability (SD = 2,220 ng Cry1Ab toxin/g fresh leaf weight, p < 0.01) was seen lengthwise along the leaf, starting at 1,892 ng Cry1Ab toxin/g fresh weight at the sheath and rising to maximum concentration at the middle of the lamella. Cry1Ab toxin content may suffer significant (SD = 2,230 ng Cry1Ab toxin/g fresh leaf weight, p < 0.01) decreases in the leaf due to necrotisation. The results indicate that the longitudinal dimension of the leaf has more significance for sampling purposes than the diagonal position.
Keywords: Genetically modified organism (GMO); MON 810 maize; Cry1Ab toxin; Enzyme-linked immunosorbent assay (ELISA)
Increased efficacy for in-house validation of real-time PCR GMO detection methods by I. M. J. Scholtens; E. J. Kok; L. Hougs; B. Molenaar; J. T. N. M. Thissen; H. van der Voet (2213-2227).
To improve the efficacy of the in-house validation of GMO detection methods (DNA isolation and real-time PCR, polymerase chain reaction), a study was performed to gain insight in the contribution of the different steps of the GMO detection method to the repeatability and in-house reproducibility. In the present study, 19 methods for (GM) soy, maize canola and potato were validated in-house of which 14 on the basis of an 8-day validation scheme using eight different samples and five on the basis of a more concise validation protocol. In this way, data was obtained with respect to the detection limit, accuracy and precision. Also, decision limits were calculated for declaring non-conformance (>0.9%) with 95% reliability. In order to estimate the contribution of the different steps in the GMO analysis to the total variation variance components were estimated using REML (residual maximum likelihood method). From these components, relative standard deviations for repeatability and reproducibility (RSDr and RSDR) were calculated. The results showed that not only the PCR reaction but also the factors ‘DNA isolation’ and ‘PCR day’ are important factors for the total variance and should therefore be included in the in-house validation. It is proposed to use a statistical model to estimate these factors from a large dataset of initial validations so that for similar GMO methods in the future, only the PCR step needs to be validated. The resulting data are discussed in the light of agreed European criteria for qualified GMO detection methods.
Keywords: In-house validation; Real-time; PCR; GMO; REML
NAIMA as a solution for future GMO diagnostics challenges by David Dobnik; Dany Morisset; Kristina Gruden (2229-2233).
In the field of genetically modified organism (GMO) diagnostics, real-time PCR has been the method of choice for target detection and quantification in most laboratories. Despite its numerous advantages, however, the lack of a true multiplexing option may render real-time PCR less practical in the face of future GMO detection challenges such as the multiplicity and increasing complexity of new transgenic events, as well as the repeated occurrence of unauthorized GMOs on the market. In this context, we recently reported the development of a novel multiplex quantitative DNA-based target amplification method, named NASBA implemented microarray analysis (NAIMA), which is suitable for sensitive, specific and quantitative detection of GMOs on a microarray. In this article, the performance of NAIMA is compared with that of real-time PCR, the focus being their performances in view of the upcoming challenge to detect/quantify an increasing number of possible GMOs at a sustainable cost and affordable staff effort. Finally, we present our conclusions concerning the applicability of NAIMA for future use in GMO diagnostics.
Keywords: Biochips; High-throughput screening; Multiplex; Genetically modified organisms; Detection; Quantification; Identification
Screening of pesticides in blood with liquid chromatography–linear ion trap mass spectrometry by Sylvain Dulaurent; Christian Moesch; Pierre Marquet; Jean-Michel Gaulier; Gérard Lachâtre (2235-2249).
In clinical or forensic toxicology, general unknown screening procedures are used to identify as many xenobiotics as possible, belonging to numerous chemical classes. We present here a general unknown screening procedure based on liquid chromatography coupled with use of a single linear ion trap mass spectrometer, and focus on the identification of pesticides and/or metabolites in whole blood. After solid-phase extraction (SPE), the compounds of interest were separated using a reversed-phase column and identified by the mass spectrometer operated first in the full-scan mass spectrometry (MS) mode, in the positive and negative polarities, followed by MS2 and MS3 scanning of ions selected in data-dependent acquisition. The total scan time was 2.45 s. Two mass spectral libraries (MS2 and MS3), each of 450 spectra, were created for the 320 pesticides and metabolites detected after injection of pure solutions. Robustness of the spectra and matrix effects were studied and were satisfactory for the present application. Detection limits for the 320 compounds were studied by extracting 1 mL spiked blood at concentrations between 10 µg/L and 10 mg/L. If necessary, it was possible to decrease the detection limits of some compounds by 10–100-fold by scanning MS2 in only one polarity, owing to a shorter total scan time. However, at the same time, the detection specificity decreased as no confirmation could be recorded in the following MS3 scan and no information could be registered in the other polarity. So, in these rare cases, confirmation by another method was required.
Keywords: General unknown screening; Pesticide; Blood; Linear ion trap
Validated assay for studying activity profiles of human liver UGTs after drug exposure: inhibition and induction studies by M. Teresa Donato; Sandra Montero; José V. Castell; M. José Gómez-Lechón; Agustín Lahoz (2251-2263).
UDP-glucuronsyltransferases (UGTs) are a family of conjugating enzymes that participate in the metabolism of many drugs. The study of potential drug–drug interactions involving UGTs has been largely hindered by the limited availability of selective functional assays for individual UGT enzymes. We propose a sensitive and reproducible procedure for the activity measurements of four major human hepatic UGT forms. The assays are based on analysis and quantification by high-performance liquid chromatography–tandem mass spectrometry of glucuronides formed from selective probe substrates, namely, β-estradiol (UGT1A1, 3-glucuronide), 1-naphthol (UGT1A6), propofol (UGT1A9), and naloxone (UGT2B7). The analytical methods developed in the present study have been validated under good laboratory practice compliance following FDA recommendations. The assays can be easily applied to both phenotyping UGT reactions in liver-derived cellular and subcellular systems, and drug–drug interaction in vitro studies. Chemical inhibition of UGTs was tested in human liver microsomes at substrate concentrations lower than the corresponding K M values. Under these conditions, selective inhibition of UGT2B7 by fluconazole and low amitriptyline concentrations were observed, whereas diclofenac and quinidine were shown as non-enzyme-selective inhibitors of UGTs. Induction of UGTs was studied in primary human hepatocytes and HepG2 cells cultured in 96-well plates. Aryl hydrocarbon receptor ligands (except indirubin in hepatocytes) increased the UGT1A1 activity in both cell models. The highest effects were observed in HepG2 cells exposed to indirubin (21-fold over the control) and omeprazole or β-naphthoflavone (about sixfold). Although variable effects were observed in other UGT enzymes, the degree of induction was generally lower than that for UGT1A1. Figure
Keywords: UDP-glucuronyltransferases; Inhibition; Induction; Liquid chromatography; Mass spectrometry
Integrated analysis of halogenated organic pollutants in sub-millilitre volumes of venous and umbilical cord blood sera by Joan O. Grimalt; Mike Howsam; Daniel Carrizo; Raquel Otero; Mary Rosa Rodrigues de Marchi; Esther Vizcaino (2265-2272).
A rapid, robust and economical method for the analysis of persistent halogenated organic compounds in small volumes of human serum and umbilical cord blood is described. The pollutants studied cover a broad range of molecules of contemporary epidemiological and legislative concern, including polychlorobiphenyls (PCBs), polychlorobenzenes (CBs), hexachlorocyclohexanes (HCHs), DDTs, polychlorostyrenes (PCSs) and polybromodiphenyl ethers (PBDEs). Extraction and clean-up with n-hexane and concentrated sulphuric acid was followed with analysis by gas chromatography coupled to electron capture (GC-ECD) and GC coupled to negative ion chemical ionisation mass spectrometry (GC-NICI-MS). The advantages of this method rest in the broad range of analytes and its simplicity and robustness, while the use of concentrated sulphuric acid extraction/clean-up destroys viruses that may be present in the samples. Small volumes of reference serum between 50 and 1000 μL were extracted and the limits of detection/quantification and repeatability were determined. Recoveries of spiked compounds for the extraction of small volumes (≥300 μL) of the spiked reference serum were between 90% and 120%. The coefficients of variation of repeatability ranged from 0.1–14%, depending on the compound. Samples of 4-year-old serum and umbilical cord blood (n = 73 and 40, respectively) from a population inhabiting a village near a chloro-alkali plant were screened for the above-mentioned halogenated pollutants using this method and the results are briefly described.
Keywords: Trace halogenated organic compounds; Pesticides; Endocrine disruptors; Blood serum; Venous serum; Umbilical cord serum; GC-MS; GC-ECD
Combination of chemical and electron-impact ionisation with GC×GC–qMS for characterization of fatty alcohol alkoxylate polymers in the low-molecular-weight range up to 700 Da by Roman Dück; Volker Wulf; Margit Geißler; Hans-Ulrich Baier; Michaela Wirtz; Hans-Willi Kling; Siegmar Gäb; Oliver J. Schmitz (2273-2283).
The class of fatty alcohol alkoxylates describes surfactants that are synthesised by reaction of fatty alcohols with alkoxides such as ethylene oxide or propylene oxide or a combination of both as copolymers. Such alkoxylates are used, for example, as nonionic surfactants in home and industrial cleaning and washing agents. Chemical characteristics of such alkoxylate copolymers, for example the degree of alkoxylation, the arrangement of building blocks (random or block polymerisation), the type of the starter, and endcapping, play an important role in application behaviour. The analysis of these characteristics is challenging because in many cases such copolymers have high polydispersity and a large number of constitutional isomers depending on the degree of alkoxylation. Furthermore, the alkoxylates often occur in a complex multicomponent matrix. Here we present a method for characterization of silylated fatty alcohol alkoxylates in the low-molecular-weight range by means of comprehensive two-dimensional gas chromatography–mass spectrometry with electron impact and chemical ionisation. This method also enables detailed analysis of the alkoxylates in a complex matrix such as modern detergents.
Keywords: GC×GC; Fatty alcohol alkoxylates; Random copolymer; Block copolymer
Experimental designs dedicated to the evaluation of a membrane extraction method: membrane-assisted solvent extraction for compounds having different polarities by means of gas chromatography–mass detection by Carole Vincelet; Jean Marc Roussel; David Benanou (2285-2292).
Membrane-assisted solvent extraction was applied for the determination of different classes of compounds in water, having K o/w (octanol–water partition coefficient) values between 101 (aniline) and 108 (methyl stearate), by means of experimental designs. Four solvents were investigated—propan-2-ol, ethyl acetate, diisopropyl ether and cyclohexane—as well as extraction time, temperature, salt impact, pH and methanol addition. The best choice was diisopropyl ether, 50 °C, 30 min and an addition of 3 g of sodium chloride at pH 2 for polar compounds. The relative standard deviation (n = 3) was found in the range from 5 to 17%. Recoveries ranged between 34 and 100%. Membrane-assisted solvent extraction was successfully applied to a fast screening method dedicated to an unknown wastewater sample. Figure Experimental setup for membrane assisted solvent extraction MASE
Keywords: Membrane-assisted solvent extraction; Extraction; Nonporous polypropylene membrane; Gas chromatography–mass spectrometry; Water analysis; Design of experiments
Correlation between lactosylation and denaturation of major whey proteins: an investigation by liquid chromatography–electrospray ionization mass spectrometry by Ilario Losito; Elisabetta Stringano; Saverio Carulli; Francesco Palmisano (2293-2306).
The Maillard-reaction-induced lactosylation of the major whey proteins, α-lactalbumin (α-La) and β-lactoglobulins (β-Lg) A and B, occurring upon heating at 70, 80 and 90 °C for 1 to 5 h in the presence of lactose excess, was studied by HPLC coupled to electrospray ionization single and tandem mass spectrometry (HPLC-ESI-MS, MS/MS). The presence of significant amounts of mono and bi-lactosylated forms of the three proteins and their increase with heating temperature and time were assessed from MS data. Evidences for a concomitant, significant denaturation, involving partial tertiary structure unfolding, were also obtained in the case of β-lactoglobulins. A subsequent ESI-MS and MS/MS investigation on the tryptic digests of heated protein solutions exhibiting high percentages of mono and bi-lactosylated forms provided information on lactosylation sites. In particular, the latter were identified both on tryptic and on aspecific peptides, whose unusual relevance (compared to similar studies) was found to be due mainly to heat-induced protein degradation, occurring before protein digestion with trypsin. Among lactosylation sites identified only on tryptic peptides, i.e., those reasonably related to intact protein lactosylation, two lysines residues were found for α-La, both located in accessible regions of its tertiary structure. In the case of β-Lg, besides three sites common to variants A and B (leucine 1, lysines 70, and 75), lysine 69 was found to be lactosylated only in variant B. Its proximity to a critical region of β-Lg tertiary structure suggests that the difference between the two variants could be ascribed to a different evolution of their conformation upon heating. Figure 3D-charts comparing the peak area percent distribution for unmodified, mono- and bi-lactosylated forms of α-La, β-LgA and β-LgB under different temperature/time conditions. As shown by the legend, colour intensification corresponds to a temperature increase.
Keywords: Whey proteins; Thermal denaturation; Maillard reaction; Lactosylation; Liquid chromatography–electrospray ionization mass spectrometry
Evaluation of a modified QuEChERS method for the extraction of pesticides from agricultural, ornamental and forestal soils by M. Asensio-Ramos; J. Hernández-Borges; L. M. Ravelo-Pérez; M. A. Rodríguez-Delgado (2307-2319).
A modified version of the QuEChERS method has been developed for the determination of a group of ten organophosphorus pesticides (i.e. ethoprofos, dimethoate, diazinon, malaoxon, chlorpyrifos-methyl, fenitrothion, malathion, chlorpyrifos, fenamiphos and phosmet) and one thiadiazine pesticide (buprofezin) in three different types of soils (forestal, ornamental and agricultural). The method was validated through linearity, recovery, precision and accuracy studies, and also by carrying out a matrix-matched calibration for the three soils owing to the existence of a strong matrix effect. Acceptable recovery values were obtained (between 45 and 96%) for all the pesticides and soils, except for malathion and malaoxon in forestal and ornamental soils, from which they could not be quantitatively extracted. Limits of detection of the whole method ranged between 0.48 and 7.78 ng/g. The method was finally applied to the determination of chlorpyrifos concentration in a treated soil for cultivation of potatoes.
Keywords: QuEChERS; Pesticides; Organophosphorus pesticides; Soils; Gas chromatography; Nitrogen–phosphorus detection; Mass spectrometry
Colorimetric and electrochemical phosphodiesterase inhibition assays for yessotoxin detection: development and comparison with LC-MS/MS by Mònica Campàs; Pablo de la Iglesia; Margarita Fernández-Tejedor; Jorge Diogène (2321-2330).
This work describes the development and applicability of two functional assays for the detection of yessotoxin (YTX), a polycyclic ether marine toxin produced by dinoflagellates. The assays are based on the interaction between this toxin and the phosphodiesterase (PDE) enzyme and the subsequent measurement of the enzyme activity by colorimetric and electrochemical methods. Firstly, several enzyme substrates were tested in order to select those able to be detected by colorimetry or electrochemistry after enzymatic hydrolysis. The substrates that provided the highest absorbance values and density currents were p-nitrophenyl phenylphosphonate and α-naphthyl phosphate, respectively. After optimisation of the experimental parameters, limits of detection of 0.8 and 0.6 µM were attained by colorimetry and electrochemistry, respectively. An inhibitory effect of YTX on the PDE activity was observed. The assays have been applied to the analysis of YTX production by Protoceratium reticulatum cultures, and results were compared with liquid chromatography–tandem mass spectrometry analysis. Figure Schematic representation of the electrochemical phosphodiesterase inhibition assay for yessotoxin detection.
Keywords: Yessotoxin (YTX); Phosphodiesterase (PDE); Inhibition assay; Protoceratium reticulatum ; Liquid chromatography–tandem mass spectrometry (LC-MS/MS)
Spectrophotometric analysis of nucleic acids: oxygenation-dependant hyperchromism of DNA by Rupak Doshi; Philip J. R. Day; Paolo Carampin; Ewan Blanch; Ian J. Stratford; Nicola Tirelli (2331-2339).
The absorbance at 260 nm (A 260) is ubiquitously used for nucleic acid quantification. We show that following oxygenation, DNA solutions experience alterations in both spectral properties (hyperchromism in the UV region, λ max 260 nm) and DNA conformation. The spectral changes caused by oxygen–DNA complexation are stable for at least several weeks at room temperature or several hours at 37 °C, but are also reversible by purging with nitrogen. Our data indicate that DNA in working solutions might already exist in the oxygen-complexed state, potentially confounding spectrophotometric analyses. Further, the presence of these complexes does not appear to impart cell toxicity in vitro or affect the biophysical functional behaviour (e.g. hybridisation) of DNA. Interestingly, our work also suggests that hybridisation could determine a release of bound oxygen, a phenomenon that could open the way to the use of such systems as oxygen carriers. Figure Oxygenation causes a marked hyperchromism in the main absorption bands of DNA (260 nm). The effect of this phenomenon on the ubiquitous A260 readings can seriously affect the determination of DNA concentration
Keywords: DNA analysis; DNA absorbance; Charge–transfer complexes; Hyperchromism; Oxygenation
On-column silver substrate synthesis and surface-enhanced Raman detection in capillary electrophoresis by Nicolae Leopold; Bernhard Lendl (2341-2348).
A new, simple, and efficient approach for on-column surface-enhanced Raman scattering (SERS) detection in capillary electrophoresis (CE) is reported. A ∼50-µm SERS substrate spot was prepared by laser-induced growth of silver particles in the 100-µm inner diameter CE capillary window or in a flow cell consisting of a 250-µm inner diameter fused silica capillary connector. For this purpose, the Raman laser was focused by a 20× objective into the detection window filled with a 0.5 mM silver nitrate and 10 mM citrate buffer solution. During the CE runs, the silver substrate spot was formed in a few seconds after the analyte injection, hence the analytes adsorbed sequentially to the silver surface when the detection window was reached, followed by desorption from the silver surface and continuing the electrophoretic migration to the capillary end. Thus, beyond migration time, valuable molecular specific information was delivered by the SERS spectra. Accurate separations and high-intensity SERS spectra are shown by CE-SERS time-dependent 3D electropherograms for the analytes rhodamine 6G, 4-(2-pyridylazo)resorcinol (PAR), PAR complex with Cu(II) and methylene blue at 0.25–25 ppm concentrations, by using 1.4–3.6 mW HeNe laser power and an acquisition time of 5 s for each spectrum. Before and after each analyte passes the detection window, clean background spectra were recorded and no memory effects perturbed the SERS detection. The silver substrate is characterized by a fast preparation rate, good reproducibility, a preparation success rate of over 95% and no mentionable influence on the electrophoretic migration time, the CE-SERS and CE-UV electropherograms being in good agreement. The successful coupling of CE and on-column SERS detection opens new perspectives for monitoring CE separations.
Keywords: CE; Raman; SERS
First results on Fe solid-phase extraction from coastal seawater using anatase TiO2 nano-particles by Christophe R. Quétel; Emilia Vassileva; Ivan Petrov; Kristina Chakarova; Konstantin I. Hadjiivanov (2349-2361).
This paper describes the application of TiO2 nano-particles (anatase form) for the solid-phase extraction of iron from coastal seawater samples. We investigated the adsorption processes by infra-red spectroscopy. We compared in batch and on-(mini)column extraction approaches (0.1 and 0.05 g TiO2 per sample, respectively), combined to external calibration and detection by inductively coupled plasma mass spectrometry at medium mass resolution. Globally, this titania phase was slightly more efficient with seawater than with ultra-pure water, although between pH 2 and pH 7, the Fe retention efficiency progressed more in ultra-pure water than in seawater (6.9 versus 4.8 times improvement). Different reaction schemes are proposed between Fe(III) species and the two main categories of titania sites at pH 2 (adsorption of [FeL x ](3 − x)+ via possibly the mediation of chlorides) and at pH 7 (adsorption of [Fe(OH)2]+ and precipitation of [Fe(OH)3]0). Under optimised conditions, the inlet system was pre-cleaned by pumping 6% HCl for ∼2 h, and the column was conditioned by aspirating ultra-pure water (1.7 g min−1) and 0.05% ammonia (0.6 g min−1) for 1 min. Then 3 g seawater sample was loaded at the same flow rate while being mixed on-line with 0.05% ammonia at 0.6 g min−1 to adjust the pH to 7. The iron retained on the oxide powder was then eluted with 3 g 6% HCl (<0.002% residual salinity in the separated samples). The overall procedural blank was 220 ± 46 (2 s, n = 16) ng Fe kg−1 (the titania was renewed in the column every 20 samples, with 2-min rinsing in between samples with 6% HCl at 1.5 g min−1). The recovery estimated from the Canadian certified reference material CASS-2 was 69.5 ± 7.6% (2 s, n = 4). Typically, the relative combined uncertainty (k = 2) estimated for the measurement of ∼1 µg Fe kg−1 (0.45 µm filtered and acidified to pH 1.5) of seawater was ∼12%. We applied our method to a similar sample, from the coastal region of the North Sea. The agreement well within stated uncertainties of our result with the value obtained independently by isotope dilution mass spectrometry further validated our method.
Keywords: Iron; Solid-phase extraction; TiO2 ; Seawater; On-line separation; Inductively coupled plasma mass spectrometry; Mechanism of retention; Infra-red spectroscopy; Uncertainty estimation; Method validation
On-column derivatization with o-phthaldialdehyde for fast determination of homocysteine in human urine by Rafał Głowacki; Kamila Borowczyk; Edward Bald; Hieronim Jakubowski (2363-2366).
A new assay for urinary homocysteine is described. The assay relies on an on-column derivatization with o-phthaldialdehyde, using a reversed-phase HPLC column and detection/quantification by fluorescence. The analysis time for reduced and total homocysteine, including sample work-up, was 5 and 13 min, respectively. Quantification limit was 25 nM.
Keywords: Urinary homocysteine; o-Phthaldialdehyde; On-column derivatization; HPLC