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

    53 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

    Fingerprint

    Ligation
    Real-Time Polymerase Chain Reaction
    Oligonucleotide Probes
    Disease Management
    Research
    Nucleic Acids
    Technology
    Polymerase Chain Reaction

    Keywords

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

    Cite this

    @article{9b86e15394e24bd09d6058b1f296b6c1,
    title = "Quantitative multiplex detection of plant pathogens using a novel ligation probe-based system coupled with universal, high-throughput real-time PCR on OpenArrays(TM)",
    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.",
    keywords = "padlock probes, phytophthora-ramorum, dna microarrays, gene analysis, quantification, amplification, biotin, assay, streptavidin, communities",
    author = "{van Doorn}, R. and M. Szemes and P.J.M. Bonants and G.A. Kowalchuk and J.F. Salles and E. Ortenberg and C.D. Schoen",
    year = "2007",
    doi = "10.1186/1471-2164-8-276",
    language = "English",
    volume = "8",
    journal = "BMC Genomics",
    issn = "1471-2164",
    publisher = "Springer Verlag",

    }

    Quantitative multiplex detection of plant pathogens using a novel ligation probe-based system coupled with universal, high-throughput real-time PCR on OpenArrays(TM). / van Doorn, R.; Szemes, M.; Bonants, P.J.M.; Kowalchuk, G.A.; Salles, J.F.; Ortenberg, E.; Schoen, C.D.

    In: BMC Genomics, Vol. 8, 276, 2007.

    Research output: Contribution to journalArticleAcademicpeer-review

    TY - JOUR

    T1 - Quantitative multiplex detection of plant pathogens using a novel ligation probe-based system coupled with universal, high-throughput real-time PCR on OpenArrays(TM)

    AU - van Doorn, R.

    AU - Szemes, M.

    AU - Bonants, P.J.M.

    AU - Kowalchuk, G.A.

    AU - Salles, J.F.

    AU - Ortenberg, E.

    AU - Schoen, C.D.

    PY - 2007

    Y1 - 2007

    N2 - 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.

    AB - 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.

    KW - padlock probes

    KW - phytophthora-ramorum

    KW - dna microarrays

    KW - gene analysis

    KW - quantification

    KW - amplification

    KW - biotin

    KW - assay

    KW - streptavidin

    KW - communities

    U2 - 10.1186/1471-2164-8-276

    DO - 10.1186/1471-2164-8-276

    M3 - Article

    VL - 8

    JO - BMC Genomics

    JF - BMC Genomics

    SN - 1471-2164

    M1 - 276

    ER -