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: | Artículo |
| Lenguaje: | Inglés |
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BMC
2019
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| Materias: | |
| Acceso en línea: | https://bmcvetres.biomedcentral.com/articles/10.1186/s12917-016-0913-3 http://hdl.handle.net/20.500.12123/4560 https://doi.org/10.1186/s12917-016-0913-3 |
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| author | Iraola, Gregorio Pérez, Ruben Betancor, Laura Marandino, Ana Morsella, Claudia Graciela Mendez, Maria Alejandra Paolicchi, Fernando Piccirillo, Alessandra Tomás, Gonzalo Velilla, Alejandra Vanesa Calleros, Lucía |
| author_browse | Betancor, Laura Calleros, Lucía Iraola, Gregorio Marandino, Ana Mendez, Maria Alejandra Morsella, Claudia Graciela Paolicchi, Fernando Piccirillo, Alessandra Pérez, Ruben Tomás, Gonzalo Velilla, Alejandra Vanesa |
| author_facet | Iraola, Gregorio Pérez, Ruben Betancor, Laura Marandino, Ana Morsella, Claudia Graciela Mendez, Maria Alejandra Paolicchi, Fernando Piccirillo, Alessandra Tomás, Gonzalo Velilla, Alejandra Vanesa Calleros, Lucía |
| author_sort | Iraola, Gregorio |
| collection | INTA Digital |
| 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 102 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. |
| format | Artículo |
| id | INTA4560 |
| institution | Instituto Nacional de Tecnología Agropecuaria (INTA -Argentina) |
| language | Inglés |
| publishDate | 2019 |
| publishDateRange | 2019 |
| publishDateSort | 2019 |
| publisher | BMC |
| publisherStr | BMC |
| record_format | dspace |
| spelling | INTA45602019-03-08T14:39:19Z 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 Graciela Mendez, Maria Alejandra Paolicchi, Fernando Piccirillo, Alessandra Tomás, Gonzalo Velilla, Alejandra Vanesa Calleros, Lucía Campylobacter fetus PCR Genética Ribosomas Experimentación en Laboratorio Genetics Ribosomes Laboratory Experimentation Reacción en Cadena de la Polimerasa 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 102 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. EEA Balcarce Fil: Iraola, Gregorio. Universidad de la República. Facultad de Ciencias. Sección Genética Evolutiva; Uruguay. Institut Pasteur Montevideo. Unidad de Bioinformática; Uruguay Fil: Pérez, Ruben. Universidad de la República. Facultad de Ciencias. Sección Genética Evolutiva; Uruguay Fil: Betancor, Laura. Universidad de la República. Facultad de Medicina. Instituto de Higiene. Departamento de Bacteriología y Virología; Uruguay Fil: Marandino, Ana. Universidad de la República. Facultad de Ciencias. Sección Genética Evolutiva; Uruguay Fil: Morsella, Claudia Graciela. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Laboratorio de Bacteriología. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; Argentina Fil: Mendez, Maria Alejandra. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Laboratorio de Bacteriología. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; Argentina Fil: Paolicchi, Fernando. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Laboratorio de Bacteriología. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; Argentina Fil: Piccirillo, Alessandra. Università degli Studi di Padova. Dipartimento di Biomedicina Comparata e Alimentazione; Italia Fil: Tomás, Gonzalo. Universidad de la República. Facultad de Ciencias. Sección Genética Evolutiva; Uruguay Fil: Velilla, Alejandra Vanesa. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce. Laboratorio de Bacteriología. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; Argentina Fil: Calleros, Lucía. Universidad de la República. Facultad de Ciencias. Sección Genética Evolutiva; Uruguay 2019-03-08T14:37:59Z 2019-03-08T14:37:59Z 2016-12 info:ar-repo/semantics/artículo info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion https://bmcvetres.biomedcentral.com/articles/10.1186/s12917-016-0913-3 http://hdl.handle.net/20.500.12123/4560 1746-6148 https://doi.org/10.1186/s12917-016-0913-3 eng info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by-nc-sa/4.0/ Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International (CC BY-NC-SA 4.0) application/pdf BMC BMC Veterinary Research 12 : 286 (2016) |
| spellingShingle | Campylobacter fetus PCR Genética Ribosomas Experimentación en Laboratorio Genetics Ribosomes Laboratory Experimentation Reacción en Cadena de la Polimerasa Iraola, Gregorio Pérez, Ruben Betancor, Laura Marandino, Ana Morsella, Claudia Graciela Mendez, Maria Alejandra Paolicchi, Fernando Piccirillo, Alessandra Tomás, Gonzalo Velilla, Alejandra Vanesa 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 | Campylobacter fetus PCR Genética Ribosomas Experimentación en Laboratorio Genetics Ribosomes Laboratory Experimentation Reacción en Cadena de la Polimerasa |
| url | https://bmcvetres.biomedcentral.com/articles/10.1186/s12917-016-0913-3 http://hdl.handle.net/20.500.12123/4560 https://doi.org/10.1186/s12917-016-0913-3 |
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