title: Rapid detection of rpoB gene mutations conferring rifampin resistance in Mycobacterium tuberculosis.
abstract: Multidrug-resistant Mycobacterium tuberculosis strains are widespread and present a challenge to effective treatment of this infection. The need for a low-cost and rapid detection method for clinically relevant mutations in Mycobacterium tuberculosis that confer multidrug resistance is urgent, particularly for developing countries. We report here a novel test that detects the majority of clinically relevant mutations in the beta subunit of the RNA polymerase (rpoB) gene that confer resistance to rifampin (RIF), the treatment of choice for tuberculosis (TB). The test, termed TB ID/R, combines a novel target and temperature-dependent RNase H2-mediated cleavage of blocked DNA primers to initiate isothermal helicase-dependent amplification of a rpoB gene target sequence. Amplified products are detected by probes arrayed on a modified silicon chip that permits visible detection of both RIF-sensitive and RIF-resistant strains of M. tuberculosis. DNA templates of clinically relevant single-nucleotide mutations in the rpoB gene were created to validate the performance of the TB ID/R test. Except for one rare mutation, all mutations were unambiguously detected. Additionally, 11 RIF-sensitive and 25 RIF-resistant clinical isolates were tested by the TB ID/R test, and 35/36 samples were classified correctly (96.2%). This test is being configured in a low-cost test platform to provide rapid diagnosis and drug susceptibility information for TB in the point-of-care setting in the developing world, where the need is acute.
authors.affiliation: Great Basin Corporation, Salt Lake City, Utah, USA.
authors.forename: Wanyuan
authors.initials: W
authors.lastname: Ao
authors.forename: Stephen
authors.initials: S
authors.lastname: Aldous
authors.forename: Evelyn
authors.initials: E
authors.lastname: Woodruff
authors.forename: Brian
authors.initials: B
authors.lastname: Hicke
authors.forename: Larry
authors.initials: L
authors.lastname: Rea
authors.forename: Barry
authors.initials: B
authors.lastname: Kreiswirth
authors.forename: Robert
authors.initials: R
authors.lastname: Jenison
mesh_terms: D000995:Antitubercular Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases; D006801:Humans; D008826:Microbial Sensitivity Tests / Q000379:methods; D020125:Mutation, Missense; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012293:Rifampin / Q000494:pharmacology*; D012680:Sensitivity and Specificity
chemical_list: D000995:Antitubercular Agents; D001426:Bacterial Proteins; C505805:rpoB protein, Mycobacterium tuberculosis; D012321:DNA-Directed RNA Polymerases; D012293:Rifampin