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Large scale multiplex PCR improves pathogen detection by DNA microarrays

BACKGROUND: Medium density DNA microchips that carry a collection of probes for a broad spectrum of pathogens, have the potential to be powerful tools for simultaneous species identification, detection of virulence factors and antimicrobial resistance determinants. However, their widespread use in m...

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Autores principales: Palka-Santini, Maria, Cleven, Berit E, Eichinger, Ludwig, Krönke, Martin, Krut, Oleg
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2009
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2631447/
https://www.ncbi.nlm.nih.gov/pubmed/19121223
http://dx.doi.org/10.1186/1471-2180-9-1
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author Palka-Santini, Maria
Cleven, Berit E
Eichinger, Ludwig
Krönke, Martin
Krut, Oleg
author_facet Palka-Santini, Maria
Cleven, Berit E
Eichinger, Ludwig
Krönke, Martin
Krut, Oleg
author_sort Palka-Santini, Maria
collection PubMed
description BACKGROUND: Medium density DNA microchips that carry a collection of probes for a broad spectrum of pathogens, have the potential to be powerful tools for simultaneous species identification, detection of virulence factors and antimicrobial resistance determinants. However, their widespread use in microbiological diagnostics is limited by the problem of low pathogen numbers in clinical specimens revealing relatively low amounts of pathogen DNA. RESULTS: To increase the detection power of a fluorescence-based prototype-microarray designed to identify pathogenic microorganisms involved in sepsis, we propose a large scale multiplex PCR (LSplex PCR) for amplification of several dozens of gene-segments of 9 pathogenic species. This protocol employs a large set of primer pairs, potentially able to amplify 800 different gene segments that correspond to the capture probes spotted on the microarray. The LSplex protocol is shown to selectively amplify only the gene segments corresponding to the specific pathogen present in the analyte. Application of LSplex increases the microarray detection of target templates by a factor of 100 to 1000. CONCLUSION: Our data provide a proof of principle for the improvement of detection of pathogen DNA by microarray hybridization by using LSplex PCR.
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spelling pubmed-26314472009-01-28 Large scale multiplex PCR improves pathogen detection by DNA microarrays Palka-Santini, Maria Cleven, Berit E Eichinger, Ludwig Krönke, Martin Krut, Oleg BMC Microbiol Methodology article BACKGROUND: Medium density DNA microchips that carry a collection of probes for a broad spectrum of pathogens, have the potential to be powerful tools for simultaneous species identification, detection of virulence factors and antimicrobial resistance determinants. However, their widespread use in microbiological diagnostics is limited by the problem of low pathogen numbers in clinical specimens revealing relatively low amounts of pathogen DNA. RESULTS: To increase the detection power of a fluorescence-based prototype-microarray designed to identify pathogenic microorganisms involved in sepsis, we propose a large scale multiplex PCR (LSplex PCR) for amplification of several dozens of gene-segments of 9 pathogenic species. This protocol employs a large set of primer pairs, potentially able to amplify 800 different gene segments that correspond to the capture probes spotted on the microarray. The LSplex protocol is shown to selectively amplify only the gene segments corresponding to the specific pathogen present in the analyte. Application of LSplex increases the microarray detection of target templates by a factor of 100 to 1000. CONCLUSION: Our data provide a proof of principle for the improvement of detection of pathogen DNA by microarray hybridization by using LSplex PCR. BioMed Central 2009-01-03 /pmc/articles/PMC2631447/ /pubmed/19121223 http://dx.doi.org/10.1186/1471-2180-9-1 Text en Copyright ©2009 Palka-Santini 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
Palka-Santini, Maria
Cleven, Berit E
Eichinger, Ludwig
Krönke, Martin
Krut, Oleg
Large scale multiplex PCR improves pathogen detection by DNA microarrays
title Large scale multiplex PCR improves pathogen detection by DNA microarrays
title_full Large scale multiplex PCR improves pathogen detection by DNA microarrays
title_fullStr Large scale multiplex PCR improves pathogen detection by DNA microarrays
title_full_unstemmed Large scale multiplex PCR improves pathogen detection by DNA microarrays
title_short Large scale multiplex PCR improves pathogen detection by DNA microarrays
title_sort large scale multiplex pcr improves pathogen detection by dna microarrays
topic Methodology article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2631447/
https://www.ncbi.nlm.nih.gov/pubmed/19121223
http://dx.doi.org/10.1186/1471-2180-9-1
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