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Quantitative multiplex detection of plant pathogens using a novel ligation probe-based system coupled with universal, high-throughput real-time PCR on OpenArrays™
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, thr...
Autores principales: | , , , , , , |
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Formato: | Texto |
Lenguaje: | English |
Publicado: |
BioMed Central|1
2007
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2064939/ https://www.ncbi.nlm.nih.gov/pubmed/17697351 http://dx.doi.org/10.1186/1471-2164-8-276 |
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author | van Doorn, Ronald Szemes, Marianna Bonants, Peter Kowalchuk, George A Salles, Joana F Ortenberg, Elen Schoen, Cor D |
author_facet | van Doorn, Ronald Szemes, Marianna Bonants, Peter Kowalchuk, George A Salles, Joana F Ortenberg, Elen Schoen, Cor D |
author_sort | van Doorn, Ronald |
collection | PubMed |
description | 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 10(4). 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. |
format | Text |
id | pubmed-2064939 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2007 |
publisher | BioMed Central|1 |
record_format | MEDLINE/PubMed |
spelling | pubmed-20649392007-11-07 Quantitative multiplex detection of plant pathogens using a novel ligation probe-based system coupled with universal, high-throughput real-time PCR on OpenArrays™ van Doorn, Ronald Szemes, Marianna Bonants, Peter Kowalchuk, George A Salles, Joana F Ortenberg, Elen Schoen, Cor D BMC Genomics Methodology Article 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 10(4). 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. BioMed Central|1 2007-08-14 /pmc/articles/PMC2064939/ /pubmed/17697351 http://dx.doi.org/10.1186/1471-2164-8-276 Text en Copyright © 2007 van Doorn et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( (http://creativecommons.org/licenses/by/2.0) ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Methodology Article van Doorn, Ronald Szemes, Marianna Bonants, Peter Kowalchuk, George A Salles, Joana F Ortenberg, Elen Schoen, Cor D Quantitative multiplex detection of plant pathogens using a novel ligation probe-based system coupled with universal, high-throughput real-time PCR on OpenArrays™ |
title | Quantitative multiplex detection of plant pathogens using a novel ligation probe-based system coupled with universal, high-throughput real-time PCR on OpenArrays™ |
title_full | Quantitative multiplex detection of plant pathogens using a novel ligation probe-based system coupled with universal, high-throughput real-time PCR on OpenArrays™ |
title_fullStr | Quantitative multiplex detection of plant pathogens using a novel ligation probe-based system coupled with universal, high-throughput real-time PCR on OpenArrays™ |
title_full_unstemmed | Quantitative multiplex detection of plant pathogens using a novel ligation probe-based system coupled with universal, high-throughput real-time PCR on OpenArrays™ |
title_short | Quantitative multiplex detection of plant pathogens using a novel ligation probe-based system coupled with universal, high-throughput real-time PCR on OpenArrays™ |
title_sort | quantitative multiplex detection of plant pathogens using a novel ligation probe-based system coupled with universal, high-throughput real-time pcr on openarrays™ |
topic | Methodology Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2064939/ https://www.ncbi.nlm.nih.gov/pubmed/17697351 http://dx.doi.org/10.1186/1471-2164-8-276 |
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