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Open Access Highly Accessed Methodology article

Quantitative multiplex detection of plant pathogens using a novel ligation probe-based system coupled with universal, high-throughput real-time PCR on OpenArrays™

Ronald van Doorn12, Marianna Szemes13, Peter Bonants1, George A Kowalchuk24, Joana F Salles15, Elen Ortenberg6 and Cor D Schoen1*

Author affiliations

1 Plant Research International B.V., Droevendaalsesteeg 1, 6708 PB, Wageningen, the Netherlands

2 NIOO-Centre for Terrestrial Ecology, P.O. Box 40, 6666 ZG, Heteren, the Netherlands

3 University of Bristol, Department of Cellular and Molecular Medicine, University Walk, Bristol, BS8 1TD, UK

4 Free University of Amsterdam, Institute of Ecological Science, De Boelelaan 1085, 1081 HV, Amsterdam, the Netherlands

5 UMR CNRS 5557- Université Lyon 1, USC INRA 1193, Microbial Ecology Centre, F-69622 Villeurbanne, France

6 BioTrove, Inc. 12 Gill Street, Woburn, MA 01801-1728, USA

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Citation and License

BMC Genomics 2007, 8:276  doi:10.1186/1471-2164-8-276

Published: 14 August 2007

Abstract

Background

Diagnostics and disease-management strategies require technologies to enable the simultaneous detection and quantification of a wide range of pathogenic microorganisms. Most multiplex, quantitative detection methods available suffer from compromises between the level of multiplexing, throughput and accuracy of quantification. Here, we demonstrate the efficacy of a novel, high-throughput, ligation-based assay for simultaneous quantitative detection of multiple plant pathogens. The ligation probes, designated Plant Research International-lock probes (PRI-lock probes), are long oligonucleotides with target complementary regions at their 5' and 3' ends. Upon perfect target hybridization, the PRI-lock probes are circularized via enzymatic ligation, subsequently serving as template for individual, standardized amplification via unique probe-specific primers. Adaptation to OpenArrays™, which can accommodate up to 3072 33 nl PCR amplifications, allowed high-throughput real-time quantification. The assay combines the multiplex capabilities and specificity of ligation reactions with high-throughput real-time PCR in the OpenArray™, resulting in a flexible, quantitative multiplex diagnostic system.

Results

The performance of the PRI-lock detection system was demonstrated using 13 probes targeting several significant plant pathogens at different taxonomic levels. All probes specifically detected their corresponding targets and provided perfect discrimination against non-target organisms with very similar ligation target sites. The nucleic acid targets could be reliably quantified over 5 orders of magnitude with a dynamic detection range of more than 104. Pathogen quantification was equally robust in single target versus mixed target assays.

Conclusion

This novel assay enables very specific, high-throughput, quantitative detection of multiple pathogens over a wide range of target concentrations and should be easily adaptable for versatile diagnostic purposes.