Cargando…
A Real-Time PCR Array for Hierarchical Identification of Francisella Isolates
A robust, rapid and flexible real-time PCR assay for hierarchical genetic typing of clinical and environmental isolates of Francisella is presented. Typing markers were found by multiple genome and gene comparisons, from which 23 canonical single nucleotide polymorphisms (canSNPs) and 11 canonical i...
Autores principales: | , , , , , |
---|---|
Formato: | Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2009
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2793073/ https://www.ncbi.nlm.nih.gov/pubmed/20027310 http://dx.doi.org/10.1371/journal.pone.0008360 |
_version_ | 1782175294309793792 |
---|---|
author | Svensson, Kerstin Granberg, Malin Karlsson, Linda Neubauerova, Vera Forsman, Mats Johansson, Anders |
author_facet | Svensson, Kerstin Granberg, Malin Karlsson, Linda Neubauerova, Vera Forsman, Mats Johansson, Anders |
author_sort | Svensson, Kerstin |
collection | PubMed |
description | A robust, rapid and flexible real-time PCR assay for hierarchical genetic typing of clinical and environmental isolates of Francisella is presented. Typing markers were found by multiple genome and gene comparisons, from which 23 canonical single nucleotide polymorphisms (canSNPs) and 11 canonical insertion-deletion mutations (canINDELs) were selected to provide phylogenetic guidelines for classification from genus to isolate level. The specificity of the developed assay, which uses 68 wells of a 96-well real-time PCR format with a detection limit of 100 pg DNA, was assessed using 62 Francisella isolates of diverse genetic and geographical origins. It was then successfully used for typing 14 F. tularensis subsp. holarctica isolates obtained from tularemia patients in Sweden in 2008 and five more genetically diverse Francisella isolates of global origins. When applied to human ulcer specimens for direct pathogen detection the results were incomplete due to scarcity of DNA, but sufficient markers were identified to detect fine-resolution differences among F. tularensis subsp. holarctica isolates causing infection in the patients. In contrast to other real-time PCR assays for Francisella, which are typically designed for specific detection of a species, subspecies, or strain, this type of assay can be easily tailored to provide appropriate phylogenetic and/or geographical resolution to meet the objectives of the analysis. |
format | Text |
id | pubmed-2793073 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2009 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-27930732009-12-22 A Real-Time PCR Array for Hierarchical Identification of Francisella Isolates Svensson, Kerstin Granberg, Malin Karlsson, Linda Neubauerova, Vera Forsman, Mats Johansson, Anders PLoS One Research Article A robust, rapid and flexible real-time PCR assay for hierarchical genetic typing of clinical and environmental isolates of Francisella is presented. Typing markers were found by multiple genome and gene comparisons, from which 23 canonical single nucleotide polymorphisms (canSNPs) and 11 canonical insertion-deletion mutations (canINDELs) were selected to provide phylogenetic guidelines for classification from genus to isolate level. The specificity of the developed assay, which uses 68 wells of a 96-well real-time PCR format with a detection limit of 100 pg DNA, was assessed using 62 Francisella isolates of diverse genetic and geographical origins. It was then successfully used for typing 14 F. tularensis subsp. holarctica isolates obtained from tularemia patients in Sweden in 2008 and five more genetically diverse Francisella isolates of global origins. When applied to human ulcer specimens for direct pathogen detection the results were incomplete due to scarcity of DNA, but sufficient markers were identified to detect fine-resolution differences among F. tularensis subsp. holarctica isolates causing infection in the patients. In contrast to other real-time PCR assays for Francisella, which are typically designed for specific detection of a species, subspecies, or strain, this type of assay can be easily tailored to provide appropriate phylogenetic and/or geographical resolution to meet the objectives of the analysis. Public Library of Science 2009-12-21 /pmc/articles/PMC2793073/ /pubmed/20027310 http://dx.doi.org/10.1371/journal.pone.0008360 Text en Svensson 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, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Svensson, Kerstin Granberg, Malin Karlsson, Linda Neubauerova, Vera Forsman, Mats Johansson, Anders A Real-Time PCR Array for Hierarchical Identification of Francisella Isolates |
title | A Real-Time PCR Array for Hierarchical Identification of Francisella Isolates |
title_full | A Real-Time PCR Array for Hierarchical Identification of Francisella Isolates |
title_fullStr | A Real-Time PCR Array for Hierarchical Identification of Francisella Isolates |
title_full_unstemmed | A Real-Time PCR Array for Hierarchical Identification of Francisella Isolates |
title_short | A Real-Time PCR Array for Hierarchical Identification of Francisella Isolates |
title_sort | real-time pcr array for hierarchical identification of francisella isolates |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2793073/ https://www.ncbi.nlm.nih.gov/pubmed/20027310 http://dx.doi.org/10.1371/journal.pone.0008360 |
work_keys_str_mv | AT svenssonkerstin arealtimepcrarrayforhierarchicalidentificationoffrancisellaisolates AT granbergmalin arealtimepcrarrayforhierarchicalidentificationoffrancisellaisolates AT karlssonlinda arealtimepcrarrayforhierarchicalidentificationoffrancisellaisolates AT neubauerovavera arealtimepcrarrayforhierarchicalidentificationoffrancisellaisolates AT forsmanmats arealtimepcrarrayforhierarchicalidentificationoffrancisellaisolates AT johanssonanders arealtimepcrarrayforhierarchicalidentificationoffrancisellaisolates AT svenssonkerstin realtimepcrarrayforhierarchicalidentificationoffrancisellaisolates AT granbergmalin realtimepcrarrayforhierarchicalidentificationoffrancisellaisolates AT karlssonlinda realtimepcrarrayforhierarchicalidentificationoffrancisellaisolates AT neubauerovavera realtimepcrarrayforhierarchicalidentificationoffrancisellaisolates AT forsmanmats realtimepcrarrayforhierarchicalidentificationoffrancisellaisolates AT johanssonanders realtimepcrarrayforhierarchicalidentificationoffrancisellaisolates |