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Dual color fluorescence in situ hybridization (FISH) assays for detecting Mycobacterium tuberculosis and Mycobacterium avium complexes and related pathogens in cultures

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dc.contributor.author Shah, Jyotsna
dc.contributor.author Weltman, Helena
dc.contributor.author Narciso, Patricia
dc.contributor.author Murphy, Christina
dc.contributor.author Poruri, Akhila
dc.contributor.author Baliga, Shrikala
dc.contributor.author Sharon, Leesha
dc.contributor.author York, Mary
dc.contributor.author Cunningham, Gail
dc.contributor.author Miller, Steve
dc.contributor.author Caviedes, Luz
dc.contributor.author Gilman, Robert
dc.contributor.author Desmond, Edward
dc.contributor.author Ramasamy, Ranjan
dc.date.accessioned 2019-01-25T15:18:33Z
dc.date.available 2019-01-25T15:18:33Z
dc.date.issued 2017
dc.identifier.uri https://hdl.handle.net/20.500.12866/4641
dc.description.abstract Two rapid dual color fluorescence in situ hybridization (FISH) assays were evaluated for detecting M. tuberculosis and related pathogens in cultures. The MN Genus-MTBC FISH assay uses an orange fluorescent probe specific for the Mycobacterium tuberculosis complex (MTBC) and a green fluorescent probe specific for the Mycobacterium and Nocardia genera (MN Genus) to detect and distinguish MTBC from other Mycobacteria and Nocardia. A complementary MTBC-MAC FISH assay uses green and orange fluorescent probes specific for the MTBC and M. avium complex (MAC) respectively to identify and differentiate the two species complexes. The assays are performed on acid-fast staining bacteria from liquid or solid cultures in less than two hours. Forty-three of 44 reference mycobacterial isolates were correctly identified by the MN Genus-specific probe as Mycobacterium species, with six of these correctly identified as MTBC with the MTBC-specific probe and 14 correctly as MAC by the MAC-specific probe. Of the 25 reference isolates of clinically relevant pathogens of other genera tested, only four isolates representing two species of Corynebacterium gave a positive signal with the MN Genus probe. None of these 25 isolates were detected by the MTBC and MAC specific probes. A total of 248 cultures of clinical mycobacterial isolates originating in India, Peru and the USA were also tested by FISH assays. DNA sequence of a part of the 23S ribosomal RNA gene amplified by PCR was obtained from 243 of the 248 clinical isolates. All 243 were confirmed by DNA sequencing as Mycobacterium species, with 157 and 50 of these identified as belonging to the MTBC and the MAC, respectively. The accuracy of the MN Genus-, MTBC-and MAC -specific probes in identifying these 243 cultures in relation to their DNA sequence-based identification was 100%. All ten isolates of Nocardia, (three reference strains and seven clinical isolates) tested were detected by the MN Genus-specific probe but not the MTBC- or MAC-specific probes. The limit of detection for M. tuberculosis was determined to be 5.1x104 cfu per ml and for M. avium 1.5x104 cfu per ml in liquid cultures with the respective MTBC- and MAC-specific probes in both the MN Genus-MTBC and MTBC-MAC FISH assays. The only specialized equipment needed for the FISH assays is a standard light microscope fitted with a LED light source and appropriate filters. The two FISH assays meet an important diagnostic need in peripheral laboratories of resource-limited tuberculosis-endemic countries. en_US
dc.language.iso eng
dc.publisher Public Library of Science
dc.relation.ispartof urn:issn:1932-6203
dc.rights info:eu-repo/semantics/restrictedAccess
dc.rights.uri https://creativecommons.org/licenses/by-nc-nd/4.0/deed.es
dc.subject Sensitivity and Specificity en_US
dc.subject In Situ Hybridization, Fluorescence en_US
dc.subject Bacillus subtilis/cytology/genetics/isolation & purification/metabolism en_US
dc.subject Corynebacterium/cytology/genetics/isolation & purification/metabolism en_US
dc.subject Fluorescent Dyes en_US
dc.subject Genes, rRNA en_US
dc.subject Microscopy, Fluorescence en_US
dc.subject Mycobacterium avium Complex/classification/cytology/genetics/isolation & purification en_US
dc.subject Mycobacterium tuberculosis/classification/cytology/genetics/isolation & purification en_US
dc.subject Nocardia/cytology/genetics/isolation & purification/metabolism en_US
dc.subject RNA, Ribosomal, 16S/genetics en_US
dc.subject RNA, Ribosomal, 23S/genetics en_US
dc.title Dual color fluorescence in situ hybridization (FISH) assays for detecting Mycobacterium tuberculosis and Mycobacterium avium complexes and related pathogens in cultures en_US
dc.type info:eu-repo/semantics/article
dc.identifier.doi https://doi.org/10.1371/journal.pone.0174989
dc.subject.ocde https://purl.org/pe-repo/ocde/ford#3.02.00 es_PE


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