Enumeration of Mycobacterium leprae using real-time PCR.
Mycobacterium leprae is not cultivable in axenic media, and direct microscopic enumeration of the bacilli is complex, labor intensive, and suffers from limited sensitivity and specificity. We have developed a real-time PCR assay for quantifying M. leprae DNA in biological samples. Primers were ident...
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ftdoajarticles:oai:doaj.org/article:f1cc2f836a1345799857046897eba22c 2023-05-15T15:09:07+02:00 Enumeration of Mycobacterium leprae using real-time PCR. Richard W Truman P Kyle Andrews Naoko Y Robbins Linda B Adams James L Krahenbuhl Thomas P Gillis 2008-01-01T00:00:00Z https://doi.org/10.1371/journal.pntd.0000328 https://doaj.org/article/f1cc2f836a1345799857046897eba22c EN eng Public Library of Science (PLoS) http://europepmc.org/articles/PMC2570796?pdf=render https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0000328 https://doaj.org/article/f1cc2f836a1345799857046897eba22c PLoS Neglected Tropical Diseases, Vol 2, Iss 11, p e328 (2008) Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 article 2008 ftdoajarticles https://doi.org/10.1371/journal.pntd.0000328 2022-12-31T14:03:40Z Mycobacterium leprae is not cultivable in axenic media, and direct microscopic enumeration of the bacilli is complex, labor intensive, and suffers from limited sensitivity and specificity. We have developed a real-time PCR assay for quantifying M. leprae DNA in biological samples. Primers were identified to amplify a shared region of the multicopy repeat sequence (RLEP) specific to M. leprae and tested for sensitivity and specificity in the TaqMan format. The assay was specific for M. leprae and able to detect 10 fg of purified M. leprae DNA, or approximately 300 bacteria in infected tissues. We used the RLEP TaqMan PCR to assess the short and long-term growth results of M. leprae in foot pad tissues obtained from conventional mice, a gene knock-out mouse strain, athymic nude mice, as well as from reticuloendothelial tissues of M. leprae-infected nine-banded armadillos. We found excellent correlative results between estimates from RLEP TaqMan PCR and direct microscopic counting (combined r = 0.98). The RLEP TaqMan PCR permitted rapid analysis of batch samples with high reproducibility and is especially valuable for detection of low numbers of bacilli. Molecular enumeration is a rapid, objective and highly reproducible means to estimate the numbers of M. leprae in tissues, and application of the technique can facilitate work with this agent in many laboratories. Article in Journal/Newspaper Arctic Directory of Open Access Journals: DOAJ Articles Arctic PLoS Neglected Tropical Diseases 2 11 e328 |
institution |
Open Polar |
collection |
Directory of Open Access Journals: DOAJ Articles |
op_collection_id |
ftdoajarticles |
language |
English |
topic |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
spellingShingle |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 Richard W Truman P Kyle Andrews Naoko Y Robbins Linda B Adams James L Krahenbuhl Thomas P Gillis Enumeration of Mycobacterium leprae using real-time PCR. |
topic_facet |
Arctic medicine. Tropical medicine RC955-962 Public aspects of medicine RA1-1270 |
description |
Mycobacterium leprae is not cultivable in axenic media, and direct microscopic enumeration of the bacilli is complex, labor intensive, and suffers from limited sensitivity and specificity. We have developed a real-time PCR assay for quantifying M. leprae DNA in biological samples. Primers were identified to amplify a shared region of the multicopy repeat sequence (RLEP) specific to M. leprae and tested for sensitivity and specificity in the TaqMan format. The assay was specific for M. leprae and able to detect 10 fg of purified M. leprae DNA, or approximately 300 bacteria in infected tissues. We used the RLEP TaqMan PCR to assess the short and long-term growth results of M. leprae in foot pad tissues obtained from conventional mice, a gene knock-out mouse strain, athymic nude mice, as well as from reticuloendothelial tissues of M. leprae-infected nine-banded armadillos. We found excellent correlative results between estimates from RLEP TaqMan PCR and direct microscopic counting (combined r = 0.98). The RLEP TaqMan PCR permitted rapid analysis of batch samples with high reproducibility and is especially valuable for detection of low numbers of bacilli. Molecular enumeration is a rapid, objective and highly reproducible means to estimate the numbers of M. leprae in tissues, and application of the technique can facilitate work with this agent in many laboratories. |
format |
Article in Journal/Newspaper |
author |
Richard W Truman P Kyle Andrews Naoko Y Robbins Linda B Adams James L Krahenbuhl Thomas P Gillis |
author_facet |
Richard W Truman P Kyle Andrews Naoko Y Robbins Linda B Adams James L Krahenbuhl Thomas P Gillis |
author_sort |
Richard W Truman |
title |
Enumeration of Mycobacterium leprae using real-time PCR. |
title_short |
Enumeration of Mycobacterium leprae using real-time PCR. |
title_full |
Enumeration of Mycobacterium leprae using real-time PCR. |
title_fullStr |
Enumeration of Mycobacterium leprae using real-time PCR. |
title_full_unstemmed |
Enumeration of Mycobacterium leprae using real-time PCR. |
title_sort |
enumeration of mycobacterium leprae using real-time pcr. |
publisher |
Public Library of Science (PLoS) |
publishDate |
2008 |
url |
https://doi.org/10.1371/journal.pntd.0000328 https://doaj.org/article/f1cc2f836a1345799857046897eba22c |
geographic |
Arctic |
geographic_facet |
Arctic |
genre |
Arctic |
genre_facet |
Arctic |
op_source |
PLoS Neglected Tropical Diseases, Vol 2, Iss 11, p e328 (2008) |
op_relation |
http://europepmc.org/articles/PMC2570796?pdf=render https://doaj.org/toc/1935-2727 https://doaj.org/toc/1935-2735 1935-2727 1935-2735 doi:10.1371/journal.pntd.0000328 https://doaj.org/article/f1cc2f836a1345799857046897eba22c |
op_doi |
https://doi.org/10.1371/journal.pntd.0000328 |
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PLoS Neglected Tropical Diseases |
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2 |
container_issue |
11 |
container_start_page |
e328 |
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