AEGiS-11IAC: Detection of rifampin conferring mutations and mycobacteria speciation using Myco GeneChip.

11th International AIDS Conference


Vancouver, British Columbia — July 7-12, 1996

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Detection of rifampin conferring mutations and mycobacteria speciation using Myco GeneChip.

Int Conf AIDS 1996 Jul 7-12; 11:218 (abstract no. Th.A.395)
Gingeras TR, Berno A, Chee M, Drenkow J; Affymetrix, Santa Clara, CA, USA. Fax: 408-481-0422.

OBJECTIVE: The development of a high density oligonucleotide array assay (Myco GeneChip assay) capable of detecting mutations conferring rifampin resistance and of identifying and differentiating Mycobacterium species present in HIV-1 infected patients.

METHODS: The nucleotide sequence of a 705 base pair region of rpoB was determined for Mycobacterium tuberculosis, M. gordonae, M. xenopi, M. chelonae, M. avium, M. intracellulare, M. kansasii, M. smegmatis, and M. scrofulaceum. A high density oligonucleotide array (Myco GeneChip assay) consisting of greater than 18,000 oligonucleotide probes based on the M. tuberculosis rpoB sequence was synthesized to detect and partially characterize the nucleotide differences found within the 705 base pairs of rpoB among the 9 Mycobacterium species. In vitro-generated fluorescently labeled RNA derived from amplicons of the 705 bp regions from each Mycobacterium species were hybridized to the Myco GeneChip assay. The resulting hybridization patterns denote all nucleotide differences between M. tuberculosis and the other Mycobacterium species.

RESULTS: The Myco GeneChip was used to genotype 10 M. tuberculosis rifampin resistant clinical isolates. Mutations previously described as conferring rifampin resistance were detected in 9 of the 10 isolates. No mutations were observed in isolate 10 within the 705 bp region analyzed. Each of the M. tuberculosis isolates was also analyzed by conventional dideoxynucleotide sequencing. A total of 25 M. tuberculosis clinical isolates has been analyzed using the Myco GeneChip assay with no nucleotide polymorphisms observed among these isolates with the exception of mutations conferring rifampin resistance. Analysis of the same 705 bp region of rpoB in 10 isolates each for the other eight Mycobacterium species reveals an average of 60-70 polymorphisms in each species compared to M. tuberculosis. Hybridization patterns of each of the Mycobacteria species on the Myco GeneChip assay is unique but highly conserved among isolates of the same species. Such patterns allow for both species and strain identification.

CONCLUSION: Analysis of the rpoB gene of Mycobacterium using high density oligonucleotide arrays (Myco GeneChip assay) permits detection of mutations conferring rifampin resistance and identification of several Mycobacteria species prevalent in HIV-1 infected patients.

Keywords: AEGIS, Rifampin, Mycobacterium, Mycobacterium tuberculosis, Antibiotics, Antitubercular, Mutation, Mycobacterium Infections, Genotype, Oligonucleotide Probes, Polymorphism (Genetics), DNA Probes, In Vitro, Human, genetics, ICA11KWDaegis,rifampin,mycobacterium,mycobacteriumtuberculosis,antibiotics,antitubercular,mutation,mycobacteriuminfections,genotype,oligonucleotideprobes,polymorphism(genetics),dnaprobes,invitro,human,genetics,ica11

960707
ThA395

Copyright © 1996 - International AIDS Society (IAS). Reproduction of this abstract (other than one copy for personal reference) must be cleared through the IAS.