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A novel real-time PCR assay for quantitative detection of Campylobacter fetus based on ribosomal sequences
BACKGROUND: Campylobacter fetus is a pathogen of major concern for animal and human health. The species shows a great intraspecific variation, with three subspecies: C. fetus subsp. fetus, C. fetus subsp. venerealis, and C. fetus subsp. testudinum. Campylobacter fetus fetus affects a broad range of...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2016
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5159996/ https://www.ncbi.nlm.nih.gov/pubmed/27978826 http://dx.doi.org/10.1186/s12917-016-0913-3 |
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author | Iraola, Gregorio Pérez, Ruben Betancor, Laura Marandino, Ana Morsella, Claudia Méndez, Alejandra Paolicchi, Fernando Piccirillo, Alessandra Tomás, Gonzalo Velilla, Alejandra Calleros, Lucía |
author_facet | Iraola, Gregorio Pérez, Ruben Betancor, Laura Marandino, Ana Morsella, Claudia Méndez, Alejandra Paolicchi, Fernando Piccirillo, Alessandra Tomás, Gonzalo Velilla, Alejandra Calleros, Lucía |
author_sort | Iraola, Gregorio |
collection | PubMed |
description | BACKGROUND: Campylobacter fetus is a pathogen of major concern for animal and human health. The species shows a great intraspecific variation, with three subspecies: C. fetus subsp. fetus, C. fetus subsp. venerealis, and C. fetus subsp. testudinum. Campylobacter fetus fetus affects a broad range of hosts and induces abortion in sheep and cows. Campylobacter fetus venerealis is restricted to cattle and causes the endemic disease bovine genital campylobacteriosis, which triggers reproductive problems and is responsible for major economic losses. Campylobacter fetus testudinum has been proposed recently based on genetically divergent strains isolated from reptiles and humans. Both C. fetus fetus and C. fetus testudinum are opportunistic pathogens for immune-compromised humans. Biochemical tests remain as the gold standard for identifying C. fetus but the fastidious growing requirements and the lack of reliability and reproducibility of some biochemical tests motivated the development of molecular diagnostic tools. These methods have been successfully tested on bovine isolates but fail to detect some genetically divergent strains isolated from other hosts. The aim of the present study was to develop a highly specific molecular assay to identify and quantify C. fetus strains. RESULTS: We developed a highly sensitive real-time PCR assay that targets a unique region of the 16S rRNA gene. This assay successfully detected all C. fetus strains, including those that were negative for the cstA gene-based assay used as a standard for molecular C. fetus identification. The assay showed high specificity and absence of cross-reactivity with other bacterial species. The analytical testing of the assay was determined using a standard curve. The assay demonstrated a wide dynamic range between 10(2) and 107 genome copies per reaction, and a good reproducibility with small intra- and inter-assay variability. CONCLUSIONS: The possibility to characterize samples in a rapid, sensitive and reproducible way makes this assay a good option to establish a new standard in molecular identification and quantification of C. fetus species. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12917-016-0913-3) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-5159996 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2016 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-51599962016-12-23 A novel real-time PCR assay for quantitative detection of Campylobacter fetus based on ribosomal sequences Iraola, Gregorio Pérez, Ruben Betancor, Laura Marandino, Ana Morsella, Claudia Méndez, Alejandra Paolicchi, Fernando Piccirillo, Alessandra Tomás, Gonzalo Velilla, Alejandra Calleros, Lucía BMC Vet Res Methodology Article BACKGROUND: Campylobacter fetus is a pathogen of major concern for animal and human health. The species shows a great intraspecific variation, with three subspecies: C. fetus subsp. fetus, C. fetus subsp. venerealis, and C. fetus subsp. testudinum. Campylobacter fetus fetus affects a broad range of hosts and induces abortion in sheep and cows. Campylobacter fetus venerealis is restricted to cattle and causes the endemic disease bovine genital campylobacteriosis, which triggers reproductive problems and is responsible for major economic losses. Campylobacter fetus testudinum has been proposed recently based on genetically divergent strains isolated from reptiles and humans. Both C. fetus fetus and C. fetus testudinum are opportunistic pathogens for immune-compromised humans. Biochemical tests remain as the gold standard for identifying C. fetus but the fastidious growing requirements and the lack of reliability and reproducibility of some biochemical tests motivated the development of molecular diagnostic tools. These methods have been successfully tested on bovine isolates but fail to detect some genetically divergent strains isolated from other hosts. The aim of the present study was to develop a highly specific molecular assay to identify and quantify C. fetus strains. RESULTS: We developed a highly sensitive real-time PCR assay that targets a unique region of the 16S rRNA gene. This assay successfully detected all C. fetus strains, including those that were negative for the cstA gene-based assay used as a standard for molecular C. fetus identification. The assay showed high specificity and absence of cross-reactivity with other bacterial species. The analytical testing of the assay was determined using a standard curve. The assay demonstrated a wide dynamic range between 10(2) and 107 genome copies per reaction, and a good reproducibility with small intra- and inter-assay variability. CONCLUSIONS: The possibility to characterize samples in a rapid, sensitive and reproducible way makes this assay a good option to establish a new standard in molecular identification and quantification of C. fetus species. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12917-016-0913-3) contains supplementary material, which is available to authorized users. BioMed Central 2016-12-15 /pmc/articles/PMC5159996/ /pubmed/27978826 http://dx.doi.org/10.1186/s12917-016-0913-3 Text en © The Author(s). 2016 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Methodology Article Iraola, Gregorio Pérez, Ruben Betancor, Laura Marandino, Ana Morsella, Claudia Méndez, Alejandra Paolicchi, Fernando Piccirillo, Alessandra Tomás, Gonzalo Velilla, Alejandra Calleros, Lucía A novel real-time PCR assay for quantitative detection of Campylobacter fetus based on ribosomal sequences |
title | A novel real-time PCR assay for quantitative detection of Campylobacter fetus based on ribosomal sequences |
title_full | A novel real-time PCR assay for quantitative detection of Campylobacter fetus based on ribosomal sequences |
title_fullStr | A novel real-time PCR assay for quantitative detection of Campylobacter fetus based on ribosomal sequences |
title_full_unstemmed | A novel real-time PCR assay for quantitative detection of Campylobacter fetus based on ribosomal sequences |
title_short | A novel real-time PCR assay for quantitative detection of Campylobacter fetus based on ribosomal sequences |
title_sort | novel real-time pcr assay for quantitative detection of campylobacter fetus based on ribosomal sequences |
topic | Methodology Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5159996/ https://www.ncbi.nlm.nih.gov/pubmed/27978826 http://dx.doi.org/10.1186/s12917-016-0913-3 |
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