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Color-coded molecular beacons for multiplex PCR screening assays
The number of different fluorescent colors that can be distinguished in a PCR screening assay restricts the number of different targets that can be detected. If only six colors can be distinguished, and the probe for each target is labeled with a unique color, then only six different targets can be...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2019
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6422326/ https://www.ncbi.nlm.nih.gov/pubmed/30883590 http://dx.doi.org/10.1371/journal.pone.0213906 |
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author | Marras, Salvatore A. E. Tyagi, Sanjay Antson, Dan-Oscar Kramer, Fred Russell |
author_facet | Marras, Salvatore A. E. Tyagi, Sanjay Antson, Dan-Oscar Kramer, Fred Russell |
author_sort | Marras, Salvatore A. E. |
collection | PubMed |
description | The number of different fluorescent colors that can be distinguished in a PCR screening assay restricts the number of different targets that can be detected. If only six colors can be distinguished, and the probe for each target is labeled with a unique color, then only six different targets can be identified. Yet, it is often desirable to identify more targets. For instance, the rapid identification of which bacterial species (if any) is present in a patient’s normally sterile blood sample, out of a long list of species, would enable appropriate actions to be taken to prevent sepsis. We realized that the number of different targets that can be identified in a screening assay can be increased significantly by utilizing a unique combination of two colors for the identification of each target species. We prepared a demonstration assay in which 15 different molecular beacon probe pairs were present, each pair specific for the same identifying sequence in the 16S ribosomal RNA gene of a different bacterial species, and each pair labeled with a unique combination of two fluorophores out of the six differently colored fluorophores that our PCR instrument could distinguish. In a set of PCR assays, each containing all 30 color-coded molecular beacons, and each containing DNA from a different bacterial species, the only two colors that arose in each real-time assay identified the species-specific target sequence that was present. Due to the intrinsic low background of molecular beacon probes, these reactions were specific and extremely sensitive, and the threshold cycle reflected the abundance of the target sequence present in each sample. |
format | Online Article Text |
id | pubmed-6422326 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2019 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-64223262019-04-02 Color-coded molecular beacons for multiplex PCR screening assays Marras, Salvatore A. E. Tyagi, Sanjay Antson, Dan-Oscar Kramer, Fred Russell PLoS One Research Article The number of different fluorescent colors that can be distinguished in a PCR screening assay restricts the number of different targets that can be detected. If only six colors can be distinguished, and the probe for each target is labeled with a unique color, then only six different targets can be identified. Yet, it is often desirable to identify more targets. For instance, the rapid identification of which bacterial species (if any) is present in a patient’s normally sterile blood sample, out of a long list of species, would enable appropriate actions to be taken to prevent sepsis. We realized that the number of different targets that can be identified in a screening assay can be increased significantly by utilizing a unique combination of two colors for the identification of each target species. We prepared a demonstration assay in which 15 different molecular beacon probe pairs were present, each pair specific for the same identifying sequence in the 16S ribosomal RNA gene of a different bacterial species, and each pair labeled with a unique combination of two fluorophores out of the six differently colored fluorophores that our PCR instrument could distinguish. In a set of PCR assays, each containing all 30 color-coded molecular beacons, and each containing DNA from a different bacterial species, the only two colors that arose in each real-time assay identified the species-specific target sequence that was present. Due to the intrinsic low background of molecular beacon probes, these reactions were specific and extremely sensitive, and the threshold cycle reflected the abundance of the target sequence present in each sample. Public Library of Science 2019-03-18 /pmc/articles/PMC6422326/ /pubmed/30883590 http://dx.doi.org/10.1371/journal.pone.0213906 Text en © 2019 Marras et al http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Marras, Salvatore A. E. Tyagi, Sanjay Antson, Dan-Oscar Kramer, Fred Russell Color-coded molecular beacons for multiplex PCR screening assays |
title | Color-coded molecular beacons for multiplex PCR screening assays |
title_full | Color-coded molecular beacons for multiplex PCR screening assays |
title_fullStr | Color-coded molecular beacons for multiplex PCR screening assays |
title_full_unstemmed | Color-coded molecular beacons for multiplex PCR screening assays |
title_short | Color-coded molecular beacons for multiplex PCR screening assays |
title_sort | color-coded molecular beacons for multiplex pcr screening assays |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6422326/ https://www.ncbi.nlm.nih.gov/pubmed/30883590 http://dx.doi.org/10.1371/journal.pone.0213906 |
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