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Diagnostic application of padlock probes—multiplex detection of plant pathogens using universal microarrays
Padlock probes (PLPs) are long oligonucleotides, whose ends are complementary to adjacent target sequences. Upon hybridization to the target, the two ends are brought into contact, allowing PLP circularization by ligation. PLPs provide extremely specific target recognition, which is followed by univ...
Autores principales: | , , , , , |
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Formato: | Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2005
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1087788/ https://www.ncbi.nlm.nih.gov/pubmed/15860767 http://dx.doi.org/10.1093/nar/gni069 |
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author | Szemes, Marianna Bonants, Peter de Weerdt, Marjanne Baner, Johan Landegren, Ulf Schoen, Cor D. |
author_facet | Szemes, Marianna Bonants, Peter de Weerdt, Marjanne Baner, Johan Landegren, Ulf Schoen, Cor D. |
author_sort | Szemes, Marianna |
collection | PubMed |
description | Padlock probes (PLPs) are long oligonucleotides, whose ends are complementary to adjacent target sequences. Upon hybridization to the target, the two ends are brought into contact, allowing PLP circularization by ligation. PLPs provide extremely specific target recognition, which is followed by universal amplification and microarray detection. Since target recognition is separated from downstream processing, PLPs enable the development of flexible and extendable diagnostic systems, targeting diverse organisms. To adapt padlock technology for diagnostic purposes, we optimized PLP design to ensure high specificity and eliminating ligation on non-target sequences under real-world assay conditions. We designed and tested 11 PLPs to target various plant pathogens at the genus, species and subspecies levels, and developed a prototype PLP-based plant health chip. Excellent specificity was demonstrated toward the target organisms. Assay background was determined for each hybridization using a no-target reference sample, which provided reliable and sensitive identification of positive samples. A sensitivity of 5 pg genomic DNA and a dynamic range of detection of 100 were observed. The developed multiplex diagnostic system was validated using genomic DNAs of characterized isolates and artificial mixtures thereof. The demonstrated system is adaptable to a wide variety of applications ranging from pest management to environmental microbiology. |
format | Text |
id | pubmed-1087788 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2005 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-10877882005-04-29 Diagnostic application of padlock probes—multiplex detection of plant pathogens using universal microarrays Szemes, Marianna Bonants, Peter de Weerdt, Marjanne Baner, Johan Landegren, Ulf Schoen, Cor D. Nucleic Acids Res Methods Online Padlock probes (PLPs) are long oligonucleotides, whose ends are complementary to adjacent target sequences. Upon hybridization to the target, the two ends are brought into contact, allowing PLP circularization by ligation. PLPs provide extremely specific target recognition, which is followed by universal amplification and microarray detection. Since target recognition is separated from downstream processing, PLPs enable the development of flexible and extendable diagnostic systems, targeting diverse organisms. To adapt padlock technology for diagnostic purposes, we optimized PLP design to ensure high specificity and eliminating ligation on non-target sequences under real-world assay conditions. We designed and tested 11 PLPs to target various plant pathogens at the genus, species and subspecies levels, and developed a prototype PLP-based plant health chip. Excellent specificity was demonstrated toward the target organisms. Assay background was determined for each hybridization using a no-target reference sample, which provided reliable and sensitive identification of positive samples. A sensitivity of 5 pg genomic DNA and a dynamic range of detection of 100 were observed. The developed multiplex diagnostic system was validated using genomic DNAs of characterized isolates and artificial mixtures thereof. The demonstrated system is adaptable to a wide variety of applications ranging from pest management to environmental microbiology. Oxford University Press 2005 2005-04-28 /pmc/articles/PMC1087788/ /pubmed/15860767 http://dx.doi.org/10.1093/nar/gni069 Text en © The Author 2005. Published by Oxford University Press. All rights reserved |
spellingShingle | Methods Online Szemes, Marianna Bonants, Peter de Weerdt, Marjanne Baner, Johan Landegren, Ulf Schoen, Cor D. Diagnostic application of padlock probes—multiplex detection of plant pathogens using universal microarrays |
title | Diagnostic application of padlock probes—multiplex detection of plant pathogens using universal microarrays |
title_full | Diagnostic application of padlock probes—multiplex detection of plant pathogens using universal microarrays |
title_fullStr | Diagnostic application of padlock probes—multiplex detection of plant pathogens using universal microarrays |
title_full_unstemmed | Diagnostic application of padlock probes—multiplex detection of plant pathogens using universal microarrays |
title_short | Diagnostic application of padlock probes—multiplex detection of plant pathogens using universal microarrays |
title_sort | diagnostic application of padlock probes—multiplex detection of plant pathogens using universal microarrays |
topic | Methods Online |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1087788/ https://www.ncbi.nlm.nih.gov/pubmed/15860767 http://dx.doi.org/10.1093/nar/gni069 |
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