Cost-meetings Eden Roc, esp may 2007

C. Session 3

Session 3: From Proof of Principle to Practical Applications
Chair and keynote speaker: James Clough

Rapporteur: Elisabeth Huber

The Latest Developments in Microarray Technology
J. Clough

Oxford Gene Technology, Yarnton Oxford, United Kingdom

[e-mail]
Oxford Gene Technology (OGT) have been pioneers in the array of Oligonucleotide microarrays since 1995 when the company was founded by Professor Sir Edwin Southern to commercialise his work in this area in the Department of Biochemistry at Oxford University.

The developments in microarrays over the last 12 years have been remarkable and OGT have licensed what has become known as the Southern Array patents to a large number of microarray companies across the world.

The up take of arrays in research has been extensive and arrays are now widely used in many areas of scientific research and are also starting to play a role in Diagnostics both in humans and other organisms.

The talk will:

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  share some insights from the early development of arrays
  
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  explore the more recent development as the density race develops
  
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  outline new application areas such as ChIP on chip and aCGH
  
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  provide some insights on the direction OGT plan to take with novel microarray formats.
  

Development and validation of a microbial diagnostic microarray for detection and identification of waterborne pathogens and indicator organisms
T. Kostić

Austrian Research Centers GmbH - ARC, Environmental Molecular Analytics, Seibersdorf, Austria

[e-mail]
A major challenge in microbial diagnostics is the parallel detection and identification of low abundance pathogens within a complex microbial community. In addition, a high specificity is required, providing robust, reliable identification at least at the species level. A microbial diagnostic microarray targeting 25 of the most relevant bacterial water pathogens and indicator organisms was developed and validated. The microarray method is based on sequence-specific end labelling of oligonucleotides and on a novel concept utilizing competitive oligonucleotide probes to improve the specificity of the assay. The potential of the microarray was tested with environmental and spiked samples. High specificity and sensitivity (< 0.1% relative abundance of the targeted pathogen in the total microbial community) as well as potential for reliable parallel detection were demonstrated. Reliability of the results was confirmed using microarray-independent approaches.
16S microarray for monitoring of plant-associated bacteria
Y. Moenne Loccoz

UMR CNRS 5557 Ecologie Microbienne, Université Lyon 1, Villeurbanne, France

[e-mail]
Many plant-colonizing bacteria have positive effects on plant growth and/or health. Their role is important to take into consideration especially in disease-suppressive soils, where these bacteria confer significant plant protection against soil-borne diseases. However, while emblematic plant-beneficial taxa have received considerable research attention (e.g. biocontrol pseudomonads), plant-beneficial strains from less-studied taxa are poorly documented, including in non-cultured taxa. In this context, we have sought to better understand the taxonomic diversity of plant-associated bacteria using a taxonomic microarray based on the 16S ribosomal gene rrs. This gene includes highly and less conserved regions, which enables to design probes targeting bacteria at various taxonomic levels, from phyla to species. The 16S rRNA-based taxonomic microarray contains 20-bp oligonucleotide probes, which can be used with rrs PCR amplicons directly obtained from plant samples. PCR may be done with universal primers, or with narrower primers if the interest is put on a particular taxonomic group. Probe validation is performed using DNA from characterized bacterial strains, and experimental results from plant samples may be compared with results obtained by the cloning-sequencing approach. The 16S microarray (up to 1400 different probes) was used to assess bacterial diversity in French soil suppressive to take-all disease of wheat (caused by Gaeumannomyces graminis var. tritici) as well as Swiss soil suppressive to black root rot of tobacco (caused by the fungus Thielaviopsis basicola). In both types of soil, the approach was useful to identify particular groups of antagonistic Pseudomonas spp. producing the antifungal compound 2,4-diacetylphloroglucinol. In addition, a broader assessment of the rhizosphere community identified a range of bacterial taxa associated with soil suppressiveness. Therefore, this taxonomic microarray approach enabled analysis of bacterial diversity associated with disease-suppressive soils, and it is expected that this tool will be useful in plant health studies.
Sanguin et al. 2006. Environmental Microbiology 8:289-307.

Sanguin et al. 2006. Applied and Environmental Microbiology 72:4302-4312.

Performance of microarrays for diagnostics of viral mixed infections.

M. Sip

University of South Bohemia, Ceske Budejovice, Czech Republic

[e-mail]
Plant viruses represent an important thread for crops and generally are present in mixed infections involving the presence of several viruses in one sample. Here we summarize the design and results of tests of a microarray for the detection of major viral potato pathogens alone and in mixed infections.

Short synthetic single-stranded oligomers (40 nt) were used as capture probes. A microchip detecting potato viruses, PVA, PVS, PVM, PVX, PVY and PLRV, in both single and mixed infections was developed and tested. The chip was also designed to distinguish between the main strains of PVY and PVS.

The design of capture probes using the concepts of hybridization in a solution without taking into account the real conditions on microarrays, in particular the presence of a charged surface requires excessive testing of the chip to validate the sequences.

The results were compared to routine testing results by ELISA. The microarray tests confirmed in all cases the presence of viruses found previously by ELISA and simultaneously found further pathogens. The presence of these additional pathogens was then confirmed by RT-PCR and sequencing. Our results show that for samples containing several pathogens a well designed microarray may become a high performance practical tool.

5. Rapporteurs Report 4

James Clough from Oxford Gene Technology (OGT) in his keynote lecture gave an overview of OGT -including its origins and how the patents it is holding have enabled it to offer its current products and services. When looking to the future he remarked that the rationale of spotting arrays might be questionable. Current application areas for spotted arrays are gene expression, array comparative Genomic Hybridisation (aCGH), ChIP on chip, methylation and mRNA.

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  Gene Expression
  
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    With new spotting technologies on the horizon, individual labs might move away from in-house array spotting as high density arrays will become more readily available at lower costs.
    
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  ChIP on chip
  
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    Looking at protein binding events at a DNA level
    
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    Requirement for good antibodies
    
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    Labelling is a major component of costs
    
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    Aims are to combine gene expression and ChIP on the same array
    
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  Array CGH
  
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    Fine mapping of copy number changes
    
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    Using DNA instead of RNA
    
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    Human -diagnostics /chromosomal abnormalities ('molecular karyoptying')
    

Other areas covered included multi sample arrays which offers great potential in microfluidics as hybridisation times can be significantly accelerated.

There are also developments towards single cell analysis (digital microarrays) with an ability to look at individual cells instead of averaging results from individual cells.

This would allow expression profiling for multiple genes in single cells and analysis of genetic integrity at single cell level.

Tanja Kostić from the Austrian Research Centers GmbH described a variety of microbial diagnostic microarrays (MDMs) developed and produced in-house.

These are used for detection and identification of pathogenic bacteria in environmental samples (high specificity and sensitivity). The approach entails using the marker gene gyrB.

Various challenges during the design process were highlighted, including certain bacteria giving false positive signals. This problem was counteracted through the introduction of competitive oligonucleotides (CO) sequences.

It was also established that labeling - and not hybridisation - is a specificity determining step.

There is potential for parallel detection of different organisms in an environment (for example waterborne bacteria, viruses and protozoa) in one assay.

Novel quantification methods such as real time on-chip PCR systems would facilitate reaching this aim.

Yvan Moenne-Loccoz from UMR CNRS/Université de Lyon gave a talk on “16S microarray for monitoring of plant-associated bacteria”.

Plant associated bacteria are poorly documented, so taxonomic microarrays based on the 16S ribosomal gene rrs were used to shed more light on this area. The taxonomic diversity of plant-associated bacteria was investigated. The main focus was on plant-beneficial rhizosphere bacteria. Taxa, potentially contributing to disease suppressiveness of soils, were successfully identified.

Through a broader assessment of the rhizosphere community, certain bacterial taxa prevalent in different types of suppressive soils were identified.

There is a possibility to assess the co-occurrence of taxa.

It is being estimated that about 10 times more probes are required to get a more detailed view, as it is more of a 'survey' at the moment.

It can be beneficial in plant health studies.

Miroslav Sip from the University of South Bohemia (Czech Republic) gave a talk on “performance of microarrays for diagnosis of viral infections”, giving insight into the challenges associated with the development of an array for testing major viral potato pathogens.

This included highlighting the intricate probe design taking into consideration aspects such as 'nearest neighbour model' (influence on binding energies of neighbouring oligos) as well as weak electrostatic interaction of surface bound DNA.

Miroslav suggested building a database for capture probes, as this could help accelerating microarray development. Successful as well as failed probes ought to be included into this database with information as to which conditions make them fail or work respectively. Sophisticated algorithms will be required.

When it comes to complex samples, several probes are necessary for reliable detection. With the array developed, practical applications are already possible, however the aim is to establish an array allowing rapid diagnosis of samples with many potential microbes.