Special Interest Group Meeting

Padlock probe technology, vision of a universal, multiplex and quantitative system for versatile applications

C.D. Schoen, M. Szemes, R. van Doorn, M. Dieho, O. Mendes, M. van den Berg, T. Prins, J. van Dijk, J. Peters, J. Bergervoet, M. Vasic, H. Zuilhof, M. Slawiak, E. Lojkowska and P.J.M. Bonants, Plant Research International (PRI) B.V., Department of Biointeractions and Plant Health, Droevendaalsesteeg 1, 6708 PB Wageningen, the Netherlands

Email: cor.schoen@wur.nl

Detecting harmful quarantine (Q) organisms in plant propagation material is necessary in order to ensure safe and sustainable agriculture. Many different detection techniques have been described, each with their own protocol, equipment, chemical reagents and expertise. If different pathogens need to be detected simultaneously, this approach is costly. The multiplicity of assays available for a specific pathogen leads to a lack of consistency among the various testing agencies in Europe and hampers standardisation.

The newest development in analysis of nucleic acids is the micro-array technology, in which different oligos can be spotted on little more than one square mm. The use of these micro-fabricated DNA analysis tools will provide the next generation of inexpensive DNA diagnostics to measure different pathogens in a massively parallel manner. To develop the micro-array technology for diagnostic purposes generic DNA/RNA generic pre-amplification methods to increase sensitivity have to be developed.

Plant Research International has recently 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 long 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 using special designs of the PLPs. Advantages of the developed PLP based diagnostic applications are: flexible and easily adaptable design, high level of specificity and multiplexing, universal downstream processing after ligation, and high-throughput format with real-time analysis. Potential fields of application are targeting of quarantine pathogens, pathogens on cultivated crop, identification of micro-organisms based on multiple motifs and/or indicator organisms for soil health status. Recently developed padlock probe-based applications are multiplex target detection and genotyping, microbial community analysis and multiplex quantitative target detection. Advantages of different design strategies in different applications of the multiplex detection system will be discussed.