An inter-laboratory validation of a real time PCR assay to measure host excretion of bacterial pathogens, particularly of Mycobacterium bovis

Advances in the diagnosis of Mycobacterium bovis infection in wildlife hosts may benefit the development of sustainable approaches to the management of bovine tuberculosis in cattle. In the present study, three laboratories from two different countries participated in a validation trial to evaluate...

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Autores: Travis, Emma R, Gaze, William H, Pontiroli, Alessandra, Sweeney, Francis P, Porter, David, Mason, Sam, Keeling, Matthew J C, Jones, Rebecca M, Sawyer, Jason, Aranaz Martín, Alicia, Castellanos Rizaldos, Elena, Cork, Jennifer, Delahay, Richard J, Wilson, Gavin J, Hewinson, R Glyn, Courtenay, Orin, Wellington, Elizabeth M H
Tipo de recurso: artículo
Fecha de publicación:2011
País:España
Institución:Universidad Complutense de Madrid (UCM)
Repositorio:Docta Complutense
Idioma:inglés
OAI Identifier:oai:docta.ucm.es:20.500.14352/45175
Acceso en línea:https://hdl.handle.net/20.500.14352/45175
Access Level:acceso abierto
Palabra clave:Veterinaria
3109 Ciencias Veterinarias
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spelling An inter-laboratory validation of a real time PCR assay to measure host excretion of bacterial pathogens, particularly of Mycobacterium bovisTravis, Emma RGaze, William HPontiroli, AlessandraSweeney, Francis PPorter, DavidMason, SamKeeling, Matthew J CJones, Rebecca MSawyer, JasonAranaz Martín, AliciaCastellanos Rizaldos, ElenaCork, JenniferDelahay, Richard JWilson, Gavin JHewinson, R GlynCourtenay, OrinWellington, Elizabeth M HVeterinaria3109 Ciencias VeterinariasAdvances in the diagnosis of Mycobacterium bovis infection in wildlife hosts may benefit the development of sustainable approaches to the management of bovine tuberculosis in cattle. In the present study, three laboratories from two different countries participated in a validation trial to evaluate the reliability and reproducibility of a real time PCR assay in the detection and quantification of M. bovis from environmental samples. The sample panels consisted of negative badger faeces spiked with a dilution series of M. bovis BCG Pasteur and of field samples of faeces from badgers of unknown infection status taken from badger latrines in areas with high and low incidence of bovine TB (bTB) in cattle. Samples were tested with a previously optimised methodology. The experimental design involved rigorous testing which highlighted a number of potential pitfalls in the analysis of environmental samples using real time PCR. Despite minor variation between operators and laboratories, the validation study demonstrated good concordance between the three laboratories: on the spiked panels, the test showed high levels of agreement in terms of positive/negative detection, with high specificity (100%) and high sensitivity (97%) at levels of 10(5) cells g(-1) and above. Quantitative analysis of the data revealed low variability in recovery of BCG cells between laboratories and operators. On the field samples, the test showed high reproducibility both in terms of positive/negative detection and in the number of cells detected, despite low numbers of samples identified as positive by any laboratory. Use of a parallel PCR inhibition control assay revealed negligible PCR-interfering chemicals co-extracted with the DNA. This is the first example of a multi-laboratory validation of a real time PCR assay for the detection of mycobacteria in environmental samples. Field studies are now required to determine how best to apply the assay for population-level bTB surveillance in wildlife.Public Library ScienceUniversidad Complutense de Madrid20112011-01-0120112011-01-01journal articlehttp://purl.org/coar/resource_type/c_6501info:eu-repo/semantics/articleapplication/pdfhttps://hdl.handle.net/20.500.14352/45175reponame:Docta Complutenseinstname:Universidad Complutense de Madrid (UCM)Inglésengopen accesshttp://purl.org/coar/access_right/c_abf2Atribución 3.0 Españahttps://creativecommons.org/licenses/by/3.0/es/info:eu-repo/semantics/openAccessoai:docta.ucm.es:20.500.14352/451752026-06-02T12:44:21Z
dc.title.none.fl_str_mv An inter-laboratory validation of a real time PCR assay to measure host excretion of bacterial pathogens, particularly of Mycobacterium bovis
title An inter-laboratory validation of a real time PCR assay to measure host excretion of bacterial pathogens, particularly of Mycobacterium bovis
spellingShingle An inter-laboratory validation of a real time PCR assay to measure host excretion of bacterial pathogens, particularly of Mycobacterium bovis
Travis, Emma R
Veterinaria
3109 Ciencias Veterinarias
title_short An inter-laboratory validation of a real time PCR assay to measure host excretion of bacterial pathogens, particularly of Mycobacterium bovis
title_full An inter-laboratory validation of a real time PCR assay to measure host excretion of bacterial pathogens, particularly of Mycobacterium bovis
title_fullStr An inter-laboratory validation of a real time PCR assay to measure host excretion of bacterial pathogens, particularly of Mycobacterium bovis
title_full_unstemmed An inter-laboratory validation of a real time PCR assay to measure host excretion of bacterial pathogens, particularly of Mycobacterium bovis
title_sort An inter-laboratory validation of a real time PCR assay to measure host excretion of bacterial pathogens, particularly of Mycobacterium bovis
dc.creator.none.fl_str_mv Travis, Emma R
Gaze, William H
Pontiroli, Alessandra
Sweeney, Francis P
Porter, David
Mason, Sam
Keeling, Matthew J C
Jones, Rebecca M
Sawyer, Jason
Aranaz Martín, Alicia
Castellanos Rizaldos, Elena
Cork, Jennifer
Delahay, Richard J
Wilson, Gavin J
Hewinson, R Glyn
Courtenay, Orin
Wellington, Elizabeth M H
author Travis, Emma R
author_facet Travis, Emma R
Gaze, William H
Pontiroli, Alessandra
Sweeney, Francis P
Porter, David
Mason, Sam
Keeling, Matthew J C
Jones, Rebecca M
Sawyer, Jason
Aranaz Martín, Alicia
Castellanos Rizaldos, Elena
Cork, Jennifer
Delahay, Richard J
Wilson, Gavin J
Hewinson, R Glyn
Courtenay, Orin
Wellington, Elizabeth M H
author_role author
author2 Gaze, William H
Pontiroli, Alessandra
Sweeney, Francis P
Porter, David
Mason, Sam
Keeling, Matthew J C
Jones, Rebecca M
Sawyer, Jason
Aranaz Martín, Alicia
Castellanos Rizaldos, Elena
Cork, Jennifer
Delahay, Richard J
Wilson, Gavin J
Hewinson, R Glyn
Courtenay, Orin
Wellington, Elizabeth M H
author2_role author
author
author
author
author
author
author
author
author
author
author
author
author
author
author
author
dc.contributor.none.fl_str_mv Universidad Complutense de Madrid
dc.subject.none.fl_str_mv Veterinaria
3109 Ciencias Veterinarias
topic Veterinaria
3109 Ciencias Veterinarias
description Advances in the diagnosis of Mycobacterium bovis infection in wildlife hosts may benefit the development of sustainable approaches to the management of bovine tuberculosis in cattle. In the present study, three laboratories from two different countries participated in a validation trial to evaluate the reliability and reproducibility of a real time PCR assay in the detection and quantification of M. bovis from environmental samples. The sample panels consisted of negative badger faeces spiked with a dilution series of M. bovis BCG Pasteur and of field samples of faeces from badgers of unknown infection status taken from badger latrines in areas with high and low incidence of bovine TB (bTB) in cattle. Samples were tested with a previously optimised methodology. The experimental design involved rigorous testing which highlighted a number of potential pitfalls in the analysis of environmental samples using real time PCR. Despite minor variation between operators and laboratories, the validation study demonstrated good concordance between the three laboratories: on the spiked panels, the test showed high levels of agreement in terms of positive/negative detection, with high specificity (100%) and high sensitivity (97%) at levels of 10(5) cells g(-1) and above. Quantitative analysis of the data revealed low variability in recovery of BCG cells between laboratories and operators. On the field samples, the test showed high reproducibility both in terms of positive/negative detection and in the number of cells detected, despite low numbers of samples identified as positive by any laboratory. Use of a parallel PCR inhibition control assay revealed negligible PCR-interfering chemicals co-extracted with the DNA. This is the first example of a multi-laboratory validation of a real time PCR assay for the detection of mycobacteria in environmental samples. Field studies are now required to determine how best to apply the assay for population-level bTB surveillance in wildlife.
publishDate 2011
dc.date.none.fl_str_mv 2011
2011-01-01
2011
2011-01-01
dc.type.none.fl_str_mv journal article
http://purl.org/coar/resource_type/c_6501
dc.type.openaire.fl_str_mv info:eu-repo/semantics/article
format article
dc.identifier.none.fl_str_mv https://hdl.handle.net/20.500.14352/45175
url https://hdl.handle.net/20.500.14352/45175
dc.language.none.fl_str_mv Inglés
eng
language_invalid_str_mv Inglés
language eng
dc.rights.none.fl_str_mv open access
http://purl.org/coar/access_right/c_abf2
Atribución 3.0 España
https://creativecommons.org/licenses/by/3.0/es/
dc.rights.openaire.fl_str_mv info:eu-repo/semantics/openAccess
rights_invalid_str_mv open access
http://purl.org/coar/access_right/c_abf2
Atribución 3.0 España
https://creativecommons.org/licenses/by/3.0/es/
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Public Library Science
publisher.none.fl_str_mv Public Library Science
dc.source.none.fl_str_mv reponame:Docta Complutense
instname:Universidad Complutense de Madrid (UCM)
instname_str Universidad Complutense de Madrid (UCM)
reponame_str Docta Complutense
collection Docta Complutense
repository.name.fl_str_mv
repository.mail.fl_str_mv
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score 15,300719

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