Multispacer sequence typing for Mycobacterium tuberculosis genotyping.

BACKGROUND: Genotyping methods developed to survey the transmission dynamics of Mycobacterium tuberculosis currently rely on the interpretation of restriction and amplification profiles. Multispacer sequence typing (MST) genotyping is based on the sequencing of several intergenic regions selected af...

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Main Authors: Zoheira Djelouadji, Catherine Arnold, Saheer Gharbia, Didier Raoult, Michel Drancourt
Format: Article
Language:English
Published: Public Library of Science (PLoS) 2008-01-01
Series:PLoS ONE
Online Access:http://europepmc.org/articles/PMC2413405?pdf=render
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author Zoheira Djelouadji
Catherine Arnold
Saheer Gharbia
Didier Raoult
Michel Drancourt
author_facet Zoheira Djelouadji
Catherine Arnold
Saheer Gharbia
Didier Raoult
Michel Drancourt
author_sort Zoheira Djelouadji
collection DOAJ
description BACKGROUND: Genotyping methods developed to survey the transmission dynamics of Mycobacterium tuberculosis currently rely on the interpretation of restriction and amplification profiles. Multispacer sequence typing (MST) genotyping is based on the sequencing of several intergenic regions selected after complete genome sequence analysis. It has been applied to various pathogens, but not to M. tuberculosis. METHODS AND FINDINGS: In M. tuberculosis, the MST approach yielded eight variable intergenic spacers which included four previously described variable number tandem repeat loci, one single nucleotide polymorphism locus and three newly evaluated spacers. Spacer sequence stability was evaluated by serial subculture. The eight spacers were sequenced in a collection of 101 M. tuberculosis strains from five phylogeographical lineages, and yielded 29 genetic events including 13 tandem repeat number variations (44.82%), 11 single nucleotide mutations (37.93%) and 5 deletions (17.24%). These 29 genetic events yielded 32 spacer alleles or spacer-types (ST) with an index of discrimination of 0.95. The distribution of M. tuberculosis isolates into ST profiles correlated with their assignment into phylogeographical lineages. Blind comparison of a further 93 M. tuberculosis strains by MST and restriction fragment length polymorphism-IS6110 fingerprinting and mycobacterial interspersed repetitive units typing, yielded an index of discrimination of 0.961 and 0.992, respectively. MST yielded 41 different profiles delineating 16 related groups and proved to be more discriminatory than IS6110-based typing for isolates containing < 8 IS6110 copies (P<0.0003). MST was successfully applied to 7/10 clinical specimens exhibiting a Cts < or = 42 cycles in internal transcribed spacer-real time PCR. CONCLUSIONS: These results support MST as an alternative, sequencing-based method for genotyping low IS6110 copy-number M. tuberculosis strains. The M. tuberculosis MST database is freely available (http://ifr48.timone.univ-mrs.fr/MST_MTuberculosis/mst).
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spelling doaj.art-e5b2b3f6bc414560b298b8d938216f932022-12-21T18:44:42ZengPublic Library of Science (PLoS)PLoS ONE1932-62032008-01-0136e243310.1371/journal.pone.0002433Multispacer sequence typing for Mycobacterium tuberculosis genotyping.Zoheira DjelouadjiCatherine ArnoldSaheer GharbiaDidier RaoultMichel DrancourtBACKGROUND: Genotyping methods developed to survey the transmission dynamics of Mycobacterium tuberculosis currently rely on the interpretation of restriction and amplification profiles. Multispacer sequence typing (MST) genotyping is based on the sequencing of several intergenic regions selected after complete genome sequence analysis. It has been applied to various pathogens, but not to M. tuberculosis. METHODS AND FINDINGS: In M. tuberculosis, the MST approach yielded eight variable intergenic spacers which included four previously described variable number tandem repeat loci, one single nucleotide polymorphism locus and three newly evaluated spacers. Spacer sequence stability was evaluated by serial subculture. The eight spacers were sequenced in a collection of 101 M. tuberculosis strains from five phylogeographical lineages, and yielded 29 genetic events including 13 tandem repeat number variations (44.82%), 11 single nucleotide mutations (37.93%) and 5 deletions (17.24%). These 29 genetic events yielded 32 spacer alleles or spacer-types (ST) with an index of discrimination of 0.95. The distribution of M. tuberculosis isolates into ST profiles correlated with their assignment into phylogeographical lineages. Blind comparison of a further 93 M. tuberculosis strains by MST and restriction fragment length polymorphism-IS6110 fingerprinting and mycobacterial interspersed repetitive units typing, yielded an index of discrimination of 0.961 and 0.992, respectively. MST yielded 41 different profiles delineating 16 related groups and proved to be more discriminatory than IS6110-based typing for isolates containing < 8 IS6110 copies (P<0.0003). MST was successfully applied to 7/10 clinical specimens exhibiting a Cts < or = 42 cycles in internal transcribed spacer-real time PCR. CONCLUSIONS: These results support MST as an alternative, sequencing-based method for genotyping low IS6110 copy-number M. tuberculosis strains. The M. tuberculosis MST database is freely available (http://ifr48.timone.univ-mrs.fr/MST_MTuberculosis/mst).http://europepmc.org/articles/PMC2413405?pdf=render
spellingShingle Zoheira Djelouadji
Catherine Arnold
Saheer Gharbia
Didier Raoult
Michel Drancourt
Multispacer sequence typing for Mycobacterium tuberculosis genotyping.
PLoS ONE
title Multispacer sequence typing for Mycobacterium tuberculosis genotyping.
title_full Multispacer sequence typing for Mycobacterium tuberculosis genotyping.
title_fullStr Multispacer sequence typing for Mycobacterium tuberculosis genotyping.
title_full_unstemmed Multispacer sequence typing for Mycobacterium tuberculosis genotyping.
title_short Multispacer sequence typing for Mycobacterium tuberculosis genotyping.
title_sort multispacer sequence typing for mycobacterium tuberculosis genotyping
url http://europepmc.org/articles/PMC2413405?pdf=render
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