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New PCR systems to confirm real-time PCR detection of Mycobacterium avium subsp. paratuberculosis

BACKGROUND: Johne's disease, a serious chronic form of enteritis in ruminants, is caused by Mycobacterium avium subsp. paratuberculosis (MAP). As the organism is very slow-growing and fastidious, several PCR-based methods for detection have been developed, based mainly on the MAP-specific gene...

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Autores principales: Herthnek, David, Bölske, Göran
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2006
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1609169/
https://www.ncbi.nlm.nih.gov/pubmed/17020599
http://dx.doi.org/10.1186/1471-2180-6-87
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author Herthnek, David
Bölske, Göran
author_facet Herthnek, David
Bölske, Göran
author_sort Herthnek, David
collection PubMed
description BACKGROUND: Johne's disease, a serious chronic form of enteritis in ruminants, is caused by Mycobacterium avium subsp. paratuberculosis (MAP). As the organism is very slow-growing and fastidious, several PCR-based methods for detection have been developed, based mainly on the MAP-specific gene IS900. However, because this gene is similar to genes in other mycobacteria, there is a need for sensitive and reliable methods to confirm the presence of MAP. As described here, two new real-time PCR systems on the IS900 gene and one on the F57 gene were developed and carefully validated on 267 strains and 56 positive clinical faecal samples. RESULTS: Our confirmatory PCR systems on IS900 were found sensitive and specific, only yielding weak false positive reactions in one strain for each system. The PCR system on F57 did not elicit any false positives and was only slightly less sensitive than our primary IS900-system. DNA from both naturally infected and spiked faeces that tested positive with our primary system could be confirmed with all new systems, except one low-level infected sample that tested negative with the F57 system. CONCLUSION: We recommend using the newly constructed DH3 PCR system on the F57 gene as the primary confirmatory test for PCR positives, but should it fail due to its lower sensitivity, the DH1 and DH2 PCR systems should be used.
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spelling pubmed-16091692006-10-14 New PCR systems to confirm real-time PCR detection of Mycobacterium avium subsp. paratuberculosis Herthnek, David Bölske, Göran BMC Microbiol Methodology Article BACKGROUND: Johne's disease, a serious chronic form of enteritis in ruminants, is caused by Mycobacterium avium subsp. paratuberculosis (MAP). As the organism is very slow-growing and fastidious, several PCR-based methods for detection have been developed, based mainly on the MAP-specific gene IS900. However, because this gene is similar to genes in other mycobacteria, there is a need for sensitive and reliable methods to confirm the presence of MAP. As described here, two new real-time PCR systems on the IS900 gene and one on the F57 gene were developed and carefully validated on 267 strains and 56 positive clinical faecal samples. RESULTS: Our confirmatory PCR systems on IS900 were found sensitive and specific, only yielding weak false positive reactions in one strain for each system. The PCR system on F57 did not elicit any false positives and was only slightly less sensitive than our primary IS900-system. DNA from both naturally infected and spiked faeces that tested positive with our primary system could be confirmed with all new systems, except one low-level infected sample that tested negative with the F57 system. CONCLUSION: We recommend using the newly constructed DH3 PCR system on the F57 gene as the primary confirmatory test for PCR positives, but should it fail due to its lower sensitivity, the DH1 and DH2 PCR systems should be used. BioMed Central 2006-10-04 /pmc/articles/PMC1609169/ /pubmed/17020599 http://dx.doi.org/10.1186/1471-2180-6-87 Text en Copyright © 2006 Herthnek and Bölske; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( (http://creativecommons.org/licenses/by/2.0) ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methodology Article
Herthnek, David
Bölske, Göran
New PCR systems to confirm real-time PCR detection of Mycobacterium avium subsp. paratuberculosis
title New PCR systems to confirm real-time PCR detection of Mycobacterium avium subsp. paratuberculosis
title_full New PCR systems to confirm real-time PCR detection of Mycobacterium avium subsp. paratuberculosis
title_fullStr New PCR systems to confirm real-time PCR detection of Mycobacterium avium subsp. paratuberculosis
title_full_unstemmed New PCR systems to confirm real-time PCR detection of Mycobacterium avium subsp. paratuberculosis
title_short New PCR systems to confirm real-time PCR detection of Mycobacterium avium subsp. paratuberculosis
title_sort new pcr systems to confirm real-time pcr detection of mycobacterium avium subsp. paratuberculosis
topic Methodology Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1609169/
https://www.ncbi.nlm.nih.gov/pubmed/17020599
http://dx.doi.org/10.1186/1471-2180-6-87
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