Cost-meetings Eden Roc, esp may 2007

Session 2b: Current Questions and Applications

Chair: Peter Bonants (COST853)

Rapporteur: Günter Adam

Keynote Speaker: Jorg Hoheisel
Advanced molecular tools for array based analyses of genome; new concepts, techniques and applications of microarray technology

J.D. Hoheisel

Division of Functional Genome Analysis, Deutsches Krebsforschungszentrum, Heidelberg, Germany

[e-mail]
Understanding of complex functional mechanisms requires global and parallel analysis of cellular processes. Microarrays are a synonym for this kind of study and in many cases the obvious platform to such end. They have already made important contributions, although the true potential that is inherent to the technology is far greater. As part of the EU-funded Integrated Project MolTools, processes were established for SNP-typing/resequencing, transcriptional analyses, single molecule detection, a quantitative analysis of protein interactions as well as cell array technologies.

www.dkfz.de/funct_genome

PADLOCK PROBE TECHNOLOGY, Vision of a Universal, Multiplex Diagnostic System for Versatile Applications
¹C. Schoen, ²M. Szemes, ¹R. van Doorn, ¹ M. Dieho, ¹O. Mendes, ¹M. van den Berg, ³T. Prins, ³J. van Dijk, ¹J. Peters, ¹J. Bergervoet, ⁴M. Vasic, ⁴H Zuilhof, ⁵M. Slawiak, ⁵E. Lojkowska, and ¹P. Bonants.

¹Plant Research International, Department of Biointeractions and Plant Health, Wageningen, The Netherlands, ²University of Bristol, Department of Pharmacology, University Walk, United Kingdom, ³RIKILT - Institute of Food Safety, Wageningen, The Netherlands, ⁴Laboratory of Organic Chemistry, Wageningen University, The Netherlands, ⁵University of Gdansk, Department of Biotechnology, Gdansk, Poland

[e-mail]

PRI recently has developed a principle for multiplex detection based on padlock probe technology which offer a means of introducing a universal step into target detection by microarrays and real-time analyses. Padlock probes (PLPs) are oligonucleotides carrying the target complementary regions at their 5' and 3' ends, which recognize adjacent sequences on the target DNA or RNA molecule. Thus, upon hybridisation, the ends of the probes get into adjacent position, and they can be joined by enzymatic ligation. Ligation occurs and the probes are circularized only when both end segments recognize correctly the target sequences. Subsequently, the target-specific products are detected by micro-array or real-time analyses.

Padlock probe based applications

- Multiplex target detection or genotyping

- Microbial community analysis

- Multiplex quantitative target detection

Advantages of PLP based diagnostic applications

- High specificity

- High level of multiplexing

- Universal downstream processing after ligation

- Flexible and easily adaptable

- High-throughput format with real-time analysis

Targeted organisms

- Quarantine pathogens

- Pathogens dependent on cultivated crop

- Identification of microorganisms based on multiple motifs

- Indicator organisms for soil health status

Use of padlock probes for detection of pectinolytic Pectobacterium carotovorum and Dickeya isolates
^aM. Slawiak, ^aE. Lojkowska, ^cM. Szemes, ^bA. Dullemans, ^bR. van Doorn, ^bA. Speksnijder, ^aM. Waleron, ^aK. Waleron, ^bP. Bonants, ^bC. D. Schoen.

^aUniversity of Gdansk, Department of Biotechnology, Gdansk, Poland, ^bPlant Research International, Department of Biointeractions and Plant Health, Wageningen, the Netherlands, ^cUniversity of Bristol, Department of Pharmacology, University Walk, Bristol, United Kingdom

[e-mail]

Pectobacterium carotovorum subsp. atrosepticum (Pca) and Pectobacterium carotovorum subsp. carotovorum (Pcc) were former described as Erwinia carotovora subsp. atroseptica and Erwnia carotovora subsp. carotovora, Dickeya spp. (Dch) was earlier described as Erwinia chrysanthemi. They are gram negative facultative anaerobe bacilli bacteria, which belong to the Gammaproteobacteria; Enterobacteriales. Dch can cause blackleg and stem rot, while Pca and Pcc cause blackleg, stem rot, soft rot symptoms in potato as well as tuber decay during storage. Early detection of Pectobacterium carotovorum and Dickeya based on DNA techniques can contribute to phytosanitary management.

Genotyping was performed on members of the former Erwinia genus. The method is based on the analysis of restriction fragment length polymorphism of the recA gene fragment amplified by PCR (recA PCR-RFLP). PCR primers, from published recA gene sequences of Pectobacterium carotovorum subsp. carotovorum, allowed the amplification of an approximately 730 bp DNA fragment from each of tested Pectobacterium and Dickeya strains. Amplified recA fragments were compared using RFLP analysis with four endonucleases (AluI, HinfI, TasI and Tru1I), allowing the detection of characteristic patterns of RFLP products for Pectobacterium and Dickeya species. Based on this analysis, diverse profiles: 2 for Pca, 19 for Pcc and 15 for Dch were presented. The phylogenetic relations of Pcc isolates with other Pectobacterium and Dickeya species based on the recA gene sequencing has been explored. A distinct subgroup of Pcc (B1), which appeared to be identical with the 3rd Polish recA PCR-RFLP profile, has been identified. The selected sub-group of Pcc as well as homogeneous sub-groups Pca and Dch was targeted for the development of a detection method as a basis for future epidemiological and ecological diagnostics.

To characterize this set of isolates, we developed an assay for its detection. We selected ligation-based detection by cleavable padlock probes (PLPs) as a method of choice because of its superior ability to discriminate minor diagnostic motifs. PLPs are long, linear oligonucleotides carrying the target complementary regions at their 5' and 3' ends. Upon hybridization, the ends of the probes get into adjacent position, and they can be joined by enzymatic ligation. Ligation occurs and the probes are circularized only when both end segments recognize the target sequences correctly. Besides the target specific recognition sequences, the PLPs contain a unique identifier sequence, the so-called ZipCode, which will hybridize to the complementary ZipCodes on the microarray. Further, a desthiobiotin moiety and a deoxy-uracyl cleavage site has been introduced for specific PLP detection.

Based on recA alignment, 4 Padlock Probes were designed: 1 for the Pcc subgroup, 1 for the Pca and 2 for Dch group. Moreover, a positive control was designed to distinguish non-target Pectobacterium and Dickeya species from false negative samples. As a target region, 16S rRNA was used. Our design criteria were to detect all Pectobacterium, Dickeya species and related bacteria, while the designed PLP should not hybridize with potato DNA.

We proved that phylogenetic probing by ligation-based detection with cleavable padlock probes is a powerful tool in the distinction of phylogenetic groups and sub-groups of Pectobacterium carotovorum subsp. atrosepticum, Pectobacterium carotovorum subsp. carotovorum and Dickeya. The method can target minimal base differences and troublesome sequence stretches in a presence of mixture of bacterial DNA and tissue from potato tubers.
Multi-detection of GMOs and plant-specific DNA in complex matrices.
T. Prins

RIKILT, Wageningen, The Netherlands

[e-mail]
Rikilt Institute of food safety has explored the padlock approach for multi-detection of (unapproved) Genetically Modified Organisms (GMOs). This approach involves ligation detection that can be done in a multiplex way, followed by universal PCR amplification of circularised products and subsequent identification on a universal microarray.

The idea behind the padlock approach is the detection and subsequent targeted amplification of DNA sequences of interest. This approach can be applied to the detection of GMO events in three ways:

• 
  by using event-specific detection methods for unapproved GMOs.
  
• 
  by using element- and/or construct-specific detection methods that recognize specific elements of GMOs already on the market or of other GMOs for which reference materials are available.
  
• 
  by using species-specific detection methods to determine the composition of the food or feed.
  

These ways will be combined in one padlock approach. If there is suspicion that unapproved GMO varieties may be present, microarray read-out may further indicate or confirm their presence. When no event-specific methods are available for the unapproved GMO varieties, the array results may form the starting-point for further investigations.

The padlock approach is based on the principle that a unique sequence is detected by a padlock probe in isolated plant DNA. Only when both ends of the padlock probe hybridize juxtaposed to their specific complementary target sequence, ligation can occur and will result in a circular molecule. After ligation, universal primer sites in the padlock probe will enable amplification of circularized padlocks. Each amplified (Cy3-labelled) padlock probe contains a unique ZIP code (a random 20 nucleotide sequence). Only amplified padlock probes will yield a signal when the pool of PCR products is hybridized to a microarray with complementary ZIP codes.

We report the identification of different (GMO-derived) crop plant products by microarray hybridization. A microarray with 50 unique cZIP-codes for the detection of padlock probes was spotted in quadruplicate. Specificity of these arrays has been tested with a pool of labelled ZIP probes as proof of principle. Furthermore, results on the testing of the methodology in relation to specificity and sensitivity will be presented, both in the analysis of single padlock probes, as well as in a multiplex setting.
Nanoparticle-Based Bio-Bar Code technology for ultra sensitive (plant) pathogen detection
O. Mendes, M. Dieho, M. Vasic*, R. van Doorn, A. van Amerongen^**, H. Zuilhof*, P. Bonants, C. Schoen^#

Plant Research International (PRI) B.V., Wageningen, The Netherlands, ^*) Laboratory of Organic Chemistry, Wageningen University, Wageningen, The Netherlands, ^**) Agrotechnology & Food Innovations B.V., Wageningen, The Netherlands

[e-mail]
High-throughput multiplexed DNA detection is important for detection of single nucleotide polymorphism and pathogens such as common infectious diseases. DNA microarrays utilize fluorescence readout and have become a core technology for the parallel interrogation of a large number of nucleic acid sequences. However, microarray technology coupled with molecular fluorophore probes has limitations; these include the necessity for target amplification, postamplification fluorophore labelling, slow binding kinetics between target sequence and capture strand, the need for multiple-laser excitation sources and complex, expensive instrumentation. The quest for improved and uniform labelling and detection methodologies has led to the development of alternative approaches such as nanoparticle labels.

Nanoparticles have been used in biotechnology over the last 4 decades as immunocytochemical probes as well as biological tags. During the last decade, however, there has been an increasing interest in using nanoparticles in DNA detection. Recent advances in functionalizing particles with oligonucleotides and tailoring their surface properties have paved the way for the development of a series of new and practical bio-detection systems. Combining new capabilities for controlling particle size and composition with versatile surface modification approaches allows for the design of optically and chemically encoded nanoparticle probes. These probes are used in biomolecule detection assays with numerous advantages when compared to conventional assays. For nucleic acid targets, all of these properties have allowed for the design of numerous assays.

In this presentation examples are reviewed and compared to the most relevant conventional techniques.
Quantitative multiplex detection of plant pathogens using PRI-lock probes and universal, ultra-high-throughput real-time PCR on OpenArrays^TM
M. Szemes, R. van Doorn, P. Bonants*, C.D. Schoen

Plant Research International B.V., The Netherlands

[e-mail]

Current technologies for multiplex, quantitative analyses frequently suffer from compromises between the level of multiplexing, throughput and accuracy of quantification. In general, for the detection of nucleic acids, microarrays provide very high level of multiplexing, but less accurate quantification and usually low throughput. At present, real-time, quantitative PCR provides the most reliable means of target quantification, and it is suitable for the analysis of a relatively high number of samples. The achievable level of multiplexing, however, is low.

Nano-scale technology, provides high-density and low-volume microchambers, which could accommodate very high number of reactions, performed under standard conditions. Many of these systems are still at the experimental phase, and are not capable of monitoring the fluorescent signals in real time for each microwell, which is required for quantification.

Recently, a conceptually new, ultra-high-throughput platform has become available for real-time PCR, capable of accommodating more than 3000 reactions per array The OpenArray^TM-s have 48 subarrays, allowing parallel testing of up to 48 samples, and each subarray contains 64 microscopic through-holes of 33 nL volume. The primers are pre-loaded into the holes, while the sample along with the reagents are auto-loaded due to surface tension, provided by the hydrophilic coating of the holes and the hydrophobic surface of the array.

Plant Research International recently has developed PRI-Lock probes for multiplex detection which provide flexibility, and bridge the gap between target-specific recognition and high-throughput amplification using universal but unique primer pairs and a generic TaqMan probe. PRI-lock probes are long oligonucleotides, similar in structure to padlock probes. They contain artificially selected primer sites and a TaqMan probe region, flanked by target complementary regions.

In this study, we have characterized the quantitation power of circularizable ligation probes, and report the development of a high-throughput, quantitative multiplex diagnostic assay based on the described principle.

Multiplex bead based detection of plant pathogens using the universal xMap technology
J.H.W. Bergervoet¹, J. Peters¹, J.M. van der Wolf¹, R. van der Vlugt¹, J.Jacobson<sup>2

1</sup> Plant Research International, Wageningen, the Netherlands, ² Luminex Corporation, Austin, United States of America

[e-mail]

The Luminex xMAP® technology was used to develop a microsphere immunoassays for multiplex detection plant pathogens. Plant samples were incubated with antibody coated fluorescent beads and with secondary antibodies, conjugated with a reporter dye. Samples were analyzed with the Luminex 100ST analyzer, in which one laser identifies individual beads and a second laser the reporter dye. These assays can be completed within 1 hour, are very robust, user-friendly, use low labor input and are suitable for high throughput screening. For potato viruses the sensitivity of the Luminex assay is approx. 10 times better when compared to standard ELISA. These microspheres can also be used for DNA or mRNA based assays.

4. Rapporteurs Remarks 3

In his keynote lecture J.D. Hoheisel showed on several different examples how microarrays help to solve nowadays complex question in molecular science and diagnostic needs mainly related to health and cancer research. He also outlined some problems related to the miniaturization involved in the array technology. As an outlook into future applications and challenges he gave first examples to image analyses of single molecules in living cells and the aim to produce in situ expressed protein chips exploiting the human genome data to express all human proteins on a chip and make them amenable for interaction and functional studies.

Cor Schön started the presentations of several Padlock probe devoted talks by first explaining the principle which is behind this family of related instruments to facilitate the array detection and quantification of nucleic acids. Starting from the original idea he introduced all modifications that have been intoduced to adjust the different derivatives for their specific tasks.

How to apply padlock probes for the identification of important bacterial pathogens of the genera Pectobacterium and Dickeya was the topic of M. Slawiak’s talk. Her studies described the research done to explore the diversity of the microbial pathogens and to identify suitable molecular markers to be used. From the selected targets (gyrB, rpos, ITS, 16 S, RecA) first amplicons were made and then were used to ligate the padlock probes and therefore make the array detection. Good results were described for RecA and the 16S rDNA.

The next talk described the possible application of padlock probes and array technology in the field of consumer protection and food quality monitoring. T. Prince described at what levels contaminations or impurities in different kinds of food products could be detected and how the GMO content could be measured according to legislative rules. As one method to improve sensitivity he suggested the cricular amplification of padlocks using Vent exo DNA polymerase which delivers ss DNA as product.

The aim to run an array detection like an ELISA test without pre-amplification of the target and/or labelling amplicons or targets with fluorophores was addressed by the subsequent talk from C. Schoen. Here the exploitation of gold or polystyren nanoparticles as carriers of the signals in sufficient strength was examined. This was applied together with a new derivative of the padlock probe BioBarCode-Padlock (BBC). To improve the fluorescent signal, helper probes were integrated which due to their neighbor effects increased the signal strength.

The possibilities to make a quantitative multiplex PCR was introduced by P.Bonants, who explained the application of the PRI-Padlock probe derivative as a modification to use the Biotrove platform for a multiplex quantitative PCR. On this platform, more than 3000 different qPCRs can be run in parallel. To improve sensitivity, a linear amplification using a rolling circle amplification with the derivate Phi-Lock of the padlock family was successfully tested,

Finally, J. Bergervoet introduced the liquid array platform from xMAP, Luminex-beads. Here, coloured plastic beads were used as surface for a sandwich ELISA protocol. Using a flow-through laser photometer, the bead type defining the antibody specificity and the labelled second antibody giving the bound quantity of antigen are determined in real time. This allows with available technology a parallel detection of 100 different targets in one microtiter well. The sensitivities as well as application of this versatile platform for nucleic acid based detection or the use of padlock probes were determined and presented.