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

R. van Doorn, M. Szemes, P.J.M. Bonants, G.A. Kowalchuk, J.F. Salles, E. Ortenberg, C.D. Schoen

    Research output: Contribution to journalArticleAcademicpeer-review

    60 Citations (Scopus)

    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.
    Original languageEnglish
    Article number276
    Number of pages14
    JournalBMC Genomics
    Volume8
    DOIs
    Publication statusPublished - 2007

    Keywords

    • padlock probes
    • phytophthora-ramorum
    • dna microarrays
    • gene analysis
    • quantification
    • amplification
    • biotin
    • assay
    • streptavidin
    • communities

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