11:50 – 12:10 Frederique Pasquer: Identification of sweet cherry incompatibility alleles by microarray
12:10 – 14:00 Lunch
14:00 – 14:20 Christiane Beckman: Cultivation-independent DNA microarray based approach
for monitoring microorganisms in soil
14:20 – 14:40 Marc Kochzius: Microarray methods used for the EU project Fish and Chip
14:40 – 15:00 Miia Lindstrom: Genomic comparison of Clostridium botulinum with DNA
15:00 – 15:20 Martin Buncek: Microarray “double-hybridization” system using LNA modified probes and magnetic nanobeads
15:20 – 15:50 Break
16:10 – 18:00 Final Management Committee Meeting
Discussion and Outlook
Contributions for Final Report and Booklet
Final COST853 Meeting: Tuesday May 22nd, 2007
COST853 - What was accomplished? J. E. Frey
Agroscope Changins-Waedenswil Research Station ACW, Laboratory for Molecular Diagnostics, Waedenswil, Switzerland
The individual research teams involved in this COST Action work in many different fields in the area of agricultural research. Among the main topics common to all members of this Action are identification of plant pathogens and of genes relevant for crop breeding. DNA-based identification of pests and pathogens was already well established before this Action was initiated. However, major problems existed because of the great diversity of pests and pathogens that had to be dealt with and the lack of harmonization with respect to the gene(s) and gene fragments best suited for diagnosis of this broad range of target organisms. Thus, methods were sought that allowed robust identification at the required taxonomic level for a wide range of organisms, that were simple and economic in use. Microarray technology seemed to have the potential to accomplish this and therefore, our Action was initiated as a technology oriented effort “…to establish and support microarray technology as a new tool for breeding, diagnosis, and high throughput screening in the field of agriculture“, as it was phrased in our Memorandum of Understanding.
At the onset of this Action only few of us had hands-on knowledge of microarray technology nor direct access to it. However, within the consortium we could offer the full set of spotters, hybridisation tools and readers as well as the required skills to all members. To accommodate the observed needs in hands-on microarray and bioinformatics training we initiated the new COST format of “STSM workshops”. Between 2003 and 2006 we conducted a series of highly successful STSM workshops totalling 43 participants. This training together with the many fruitful contacts within our Action may have contributed to the significant increase in progress that could be observed in this field as our Action went on. Much effort went into the development of specific techniques, of probe design and of strategies for target labelling and many interesting achievements were first communicated at some of our Working Group Meetings. Many new contacts were established in the course of this Action and we now have a dense network to companies and to other research organisations, for example via several projects of FP6 of the European Union. In this talk I will give a short overview on some of the highlights, successes and problems encountered in the course of COST Action 853.
Array detection of tobamoviruses: proposal for general primers and specific oligonucleotides G. Adam and P. Willingmann
BioCenter Klein Flottbek, University Hamburg, Germany
Tobamoviruses can be sorted into 3 or 4 clusters which is partially based on host range, serological crossreactions, genome organisation and mainly CP nucleic acid sequence comparison. We have developed general primers for the coat protein region, however, it was necessary to use primers for at least three different clusters. In addition we have developed and tested reverse primers for the different species of the solanaceous and cucurbit cluster. These were tested for their specificity by nested PCR in all combinations and in silico for their applicability as capture oligonucleotides in an array approach. For the two clusters, where for each species several isolates were tested, the species specific primers were in all cases specific.
Microarray for detection of fruit-tree viruses: the developing and testing. O. Lenz
Biology centre ASCR, Institute of Plant Molecular Biology, Ceske Budejovice, Czech Republic
An oligonucleotide microarray was developed and tested for detection of seven fruit-tree viruses: Apple mosaic virus (ApMV), Apple chlorotic leaf spotvirus (ACLSV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV), Prune dwarf virus (PDV), Prunus necrotic ringspot virus (PNRSV) and Plum pox virus (PPV). The sensitivity of the whole microarray was assessed by anti-sense Cy3-labeled oligonucleotides; the detection limit was approximately 108 molecules of a target bearing one molecule of dye. Positively tested natural samples were used for preparation of both cDNA and PCR-amplified targets, but only the latter were detectable by the microarray. Subsequent real-time PCR of the cDNA targets revealed, that their concentration was at least 2 orders lower than the sensitivity of the microarray.
Three different Cy3/Cy5 labeling methods were compared: a post-labeling of amino-allyl dUTPs, a Cy3-primer labeling and the direct labeling by Cy5-labeled dUTPs. All the differently labeled targets hybridized to all relevant probes, except of PDV targets hybridizing to three of four relevant probes only. Considering the cross-hybridization, the most reliable method was the post-labeling, where no cross-hybridization appeared. An occasional cross-hybridization occurred with the two other methods. The highest signal-to-noise ratio showed the direct labeling, which – however – produced the weakest signal. The highest sensitivity attained the Cy3-primer labeling. Sensitivity of the post-labeling was lower than or equal to the Cy3-primer labeling. A method of the simplest preparation and the lowest cost was the Cy3-primer labeling.
Virus detection- an array of applications N. Boonham
CSL, York, United Kingdom
Microarray methods are well suited to screening plant and other material for virus infection. Unlike other pathogens (and pests) viruses do not contain conserved sequences that can be amplified using PCR for detection and identification. In the case of fungal and bacterial pathogens (ITS or 16S) and invertebrate pests (COX) conserved regions of sequence can be amplified using PCR; following sequencing a database of sequence can be interrogated to identify the species. In the case of viruses since no similar 'universal' sequences are available screening is done by performing tests in parallel - something microarray techniques were initially developed to achieve.
Potato virus Y – its diagnostics and biological impact studied by cDNA microarrys P. Kogovšek1, M. Pompe-Novak1, Š. Baebler1, H. Krečič-Stres1, A. Rotter1, L. Gow2, K. Gruden1, N. Boonham3, G.D. Foster2, M. Ravnikar1
1National Institute of Biology, Ljubljana, Slovenia, 2School of Biological Sciences, University of Bristol, United Kingdom, 3Central Science Laboratory, York, United Kingdom
Potato virus Y (PVY) is one of the most important potato viruses spread all over the world. Following the biological characteristics, PVY isolates are classified into three strains – PVYN, PVYO and PVYC, causing different types of biological reaction in different potato cultivars. In last decades new strain varieties emerged, such as PVYZ, PVYN-Wilga, PVYN:O and PVYNTN where the classification and biological characteristics are not in correlation. Since different isolates cause diverse symptoms, PVYNTN causing the most serious ones, good determination methods are crucial. Numerous serological and molecular methods have been developed to distinguish PVY isolates, but differentiation of PVYN and PVYNTN still presents a problem. That is why we propose a new real-time PCR assay which enables detection of all PVY isolates and simultaneous discrimination of PVYNTN and PVYN group of isolates.
Besides, potato plant response to different PVY isolates was studied at transcriptional level by cDNA microarrays. We optimized some steps in gene expression quantification using cDNA microarrays, including total RNA isolation, genomic DNA degradation, cDNA synthesis and data analysis. Several computer programs and statistical packages were used in data analysis. ArrayPro, R, MEV and MapMan turned out to be the most useful ones for analyzing and visualizing large data sets obtained with TIGR 10000 clones potato cDNA microarrays.
According to our preliminary results, cultivars Igor and Nadine have different patterns in expression of genes involved in starch and sucrose metabolism. Genes in both pathways appear to be up- or down-regulated in plants inoculated with PVYNTN in comparison to the response in plants inoculated with PVYN. Up-regulation of fructokinase implicates accumulation of fructose-6P in leaves inoculated with PVYNTN relative to the response in plants inoculated with PVYN. Genes for the enzymes involved in sucrose and starch synthesis, degradation and transport have a possible role in symptoms development.
Made4: a software package for multivariate analysis of gene expression data G. Perrière
Laboratoire de Biométrie et Biologie Évolutive, Université Claude Bernard – Lyon, Villeurbanne, France
Made4 is an R package devoted to multivariate analysis of microarray gene-expression data. Its originality, relatively to the large number of software available in the field, is the integration of original methods for supervised classification and cross-platform comparisons. Made4 accepts a wide variety of gene-expression data formats. Also, it takes advantage of the extensive graphical functions from another R package – ade4, extending these for application to microarray data.
Our first aim, in writing made4, was to provide a simple-to-use tool for multivariate analysis of microarray data. Multivariate approaches have been frequently applied in the analysis of microarray data. For instance, Principal Component Analysis (PCA) has been shown to be useful in exploratory analysis of linear trends in data. Although multivariate methods are available in several R packages, including stats and amap, the ade4 package contains many improvements, including methods for analysis of one-data matrix, coupling of two-data matrices or multi-table analysis. These latter methods for integrating multiple datasets make ade4 very attractive for analysis of microarray data. Therefore, made4 was developed as an extension to ade4 to facilitate input and analysis of microarray data. It is integrated in Bioconductor as an official package (http://www.bioconductor.org).
Among the original methods that can be found in made4 is the Between-Group Analysis (BGA), which is a supervised classification method. The basis of BGA is to ordinate the groups rather than the individual samples. In tests on two microarray gene-expression datasets, BGA performs comparably to a range of supervised classification methods, including support vector machines and artificial neural networks. An attractive feature of BGA is that it is not limited by the large number of genes relative to the number of samples typical of microarray data. The projection of test data on BGA axes can be performed, as well as Leave-One-Out Cross Validation (LOOCV).
Another original method is Co-Inertia Analysis (CIA). CIA is a multivariate method that identifies trends or co-relationships in multiple datasets, which contain the same cases or variables. That is, either the rows or the columns of a matrix must be “matchable”. It is similar to the partial least square (PLS) regression method and, like PLS, it can be used when the number of variables is greater than the number of observations, which is usually the case with gene expression data. We already applied CIA to the cross-platform comparison of microarray gene-expression datasets and to determine which motifs, from a database of promoter motifs, are strongly associated with differences in gene expression levels.
At last, there are many functions in made4 to visualise results. Microarray samples (or genes) can be colour coded if a vector of class membership is given. In addition, there are functions for drawing 1D and 3D plots. For example, the function html3D produces output that can be visualized using different helper applications, providing a free and very useful interface for colouring, rotating, zooming and manipulating 3D graphs.
Identification of sweet cherry incompatibility alleles by microarray F. Pasquer1, B. Frey1, T. Sonneveld2, K. Tobutt2, M. Schuster3 And J. E. Frey1
1: Agroscope Changins-Waedenswil Research Station ACW, Waedenswil, Switzerland, 2: East Malling Research, United Kingdom, 3: Institut für Obstzüchtung, Dresden, Germany
We have developed a microarray for identification of cherry incompatibility alleles. Using intron sequence information of the S-RNase genes, we have created a microarray chip that allows to specifically recognize the incompatibility alleles present in a cultivar. Most of the designed probes showed high specificity towards their alleles. In the original set of probes cross-hybridisation was observed between few alleles with high sequence homology. As our identification system is based on the combined hybridisation information from both introns, we were able to identify false positive and unspecific probes which could be eliminated from our microarray. The optimized microarray was tested with cultivars with known alleles. The chip correctly identified all tested alleles. Furthermore, it was also able to identify alleles in other cultivars where, so far, only one allele was determined. Our results demonstrate the great promise of microarray technology for this novel application.
Fish & Chips: Developing DNA microarrays for the identification of fishes, invertebrates, and phytoplankton from European seas D. Blohm & M. Kochzius
Centre for Applied Gene Sensor Technology (CAG), University of Bremen, Germany
Currently, DNA chips are almost exclusively used as research tools for gene expression analysis. Their use for the identification of organisms is not yet widely distributed and only a few studies on mammals, bacteria, and viruses have been reported. The “Fish & Chips” project aims to demonstrate that DNA chips can be a new innovative tool for the identification of marine animals and phytoplankton. To achieve this goal, biological material was sampled in European seas and fragments of the mitochondrial 16S rRNA, COI, and cyt b genes, and nuclear 18S and 28S genes were sequenced as molecular markers to enable probe design for the microarray. To share the data, an on-line data base containing all sequences and relevant information of the samples has been implemented. The “Fish Chip” under development is based on mitochondrial 16S, COI, and cyt b sequences. The 16S probes are based on 406 sequences from 50 fish species and capture oligonucleotides for 46 species could be designed. These probes are tested with 39 samples from 23 target species so far, showing very specific binding to the corresponding probe. Only a few probes show weak false-positive signals or are only specific for a certain group of fishes (e.g. family level). The “Invertebrate Chip” comprises capture oligonucleotides of 15 species. It is based on 70 16S sequences from 15 species and 149 COI sequences from five species. This microarray was tested with 30 16S samples from 15 target species and ten COI samples from five target species. The targets tested so far show very specific binding to the corresponding probe. Only very few probes are showing weak false positive signals. Probes for two species showed neither specific nor unspecific binding. A “Phytoplankton Chip” based on 18S and 28S sequences is now available with probes for all microalgal classes and many toxic species. The results we obtained for the three DNA microarrays under development show that this approach is seemingly feasible and that the genetic identification of marine organisms by this method is a realistic approach. The reliability under routine application is still under investigation
Cultivation-independent DNA Microarray Based Approach for Monitoring Micro-organisms in Soil C. Beckmann, G. Vogel, E.Kauffmann*, G. M. Kresbach*, U. Vögeli
Kantonales Laboratorium Basel-Stadt, Kontrollstelle für Chemie- und Biosicherheit, Basel, Switzerland; * Zeptosens, A Division of Bayer (Schweiz) AG, Witterswil, Switzerland
The use of pathogenic and genetically modified organisms requires appropriate containment measures to ensure public and environmental safety. As an enforcement agency for biotechnology safety guidelines we inspect biotech and research facilities and are engaged in bio-emergency and disaster response. For this purpose, we establish versatile methods for the rapid identification and quantification of bacteria and viruses in laboratories and in the environment.
The microarray technology is a widely established tool for investigation and monitoring of gene expression, but is also used more and more for DNA-sequence based identification of microorganisms in routine diagnostics or complex environments like soil, sediment or water. The quality and sensitivity of the microarrays and readout system as well as the sample handling and cleanup processes is of key importance for their successful use, especially when complex matrices are involved.
Here we report the development of a highly sensitive cultivation-independent DNA microarray-based screening method that allows the simultaneous detection and identification of a relevant set of microorganisms from crude soil samples.
We designed and validated a planar waveguide technology (PWG) based microarray approach comprising 60 probes, which target 16S rRNA genes of about 40 bacterial species relevant for biotech industry, human pathogens, potential bioterror agents and soil microorganisms. The capture probes consist of a constant part and a variable part of 24 to 33 bp with a total length of 50 bp. The oligonucleotide target sequences are labelled using the sequence specific end-labeling (SSEL§) method. Parameters were optimized by applying
multiplex SSEL for simultaneous detection of numerous bacteria.
Probe specificity was validated with 16S rDNA gene PCR products of pure bacterial cultures. In addition, spiking experiments with individual and mixed bacterial cultures were performed to demonstrate the potential of screening for the presence of different microorganisms within soil samples and for validation of the detection system.
The assay sensitivity and performance was determined by comparing microarray data with those obtained by Real Time PCR.
Using this novel approach we could show that PWG based technology in combination with SSEL is able to detect microorganisms in the range of 25 to a few hundred genome copies. Thus, while providing high-throughput and simultaneous detection, the sensitivity of the microarray is only in the range of 5- to 50-fold less sensitive than TaqMan PCR.
In summary this microbial diagnostic microarray is, potentially, a very useful tool to screen for the presence or absence of different microorganisms within soil samples.
§ Rudi, K., Treimo, J., Nissen, H., Vegarud, G. (2003), Scientific World Journal 3: 578-584
Genomic comparison of Clostridium botulinum with DNA microarrays M. Lindström1, K. Kiviniemi1, K. Hinderink1, P. Somervuo1, M. Nevas1, P. Auvinen2 & H. Korkeala1
1Department of Food and Environmental Hygiene, University of Helsinki, Finland
2 Institute of Biotechnology, University of Helsinki, Finland
Clostridium botulinum is a dangerous food pathogen that produces potentially lethal, paralytic neurotoxin during its growth. Based on their physiology, C. botulinum strains are divided into four distinct groups I-IV, with groups I (proteolytic) and II (nonproteolytic) being human pathogens and producing neurotoxin types A, B, E, or F. In this study, a genomic comparison with DNA microarrays, based on the genome of the group I type A strain ATCC 3502 (M. Peck, Institute of Food Research, Norwich, UK), of 85 C. botulinum strains from various sources was conducted. A preliminary data analysis showed significant differences between groups I and II C. botulinum, confirming the previous phylogenetic studies that suggest distinct genetic background of the two groups. Within C. botulinum group I, strains of the same toxin type were mainly grouped together. Moreover, two large clusters of nearly identical group I type B strains of Nordic origin were observed. These two clusters differed by approximately 100 genes related to e.g. arsenic and cadmium resistance. Corresponding differences were observed in physiological tests in nutrient broth. A more detailed analysis of the results is ongoing.
Microarray “double-hybridization” system using LNA modified probes and magnetic nanobeads M. Buncek
GENERI BIOTECH s.r.o., Hradec Králové, Czech Republic
To enhance the sensitivity of microarray analyses of low abundant species of nucleic acid molecules, the „double hybridization“ system utilizing LNA-containing probes attached to magnetic nano-beads for selective extraction and subsequent microarray analysis was developed.
In this study, a set of highly specific DNA-LNA probe pairs were defined suitable for detecting of five common pathogens: Staphylococcus epidermidis, Staphylococcus aureus, Listeria monocytogenes, Salmonella entericasubsp. enterica serovar Typhimurium LT2, and Salmonella enterica subsp. enterica serovar Typhi Ty2. The assay used to test the quality of the oligonucleotide pairs relies on PCR amplification of specific sequence domains of the five target pathogens as a first step, followed by selecting suitable DNA and LNA probes and using them for hybridization and cross-hybridization experiments on a chip. Preliminary results from the experiments when labelled target DNA molecules were successfully extracted by LNA-nanobeads and detected on the microarray will be presented. The results obtained showed that using double hybridization of the sequence-specific LNA and DNA probes is feasible and helpful to increase sensitivity of microarray experiments.