Evaluation of the invader assay, a linear signal amplification method, for identification of mutations associated with resistance to rifampin and isoniazid in Mycobacterium tuberculosis.
We evaluated a recently described linear signal amplification method for sensitivity and specificity in detecting mutations associated with resistance to rifampin (RIF) and isoniazid (INH) in Mycobacterium tuberculosis. The assay utilizes the thermostable flap endonuclease Cleavase VIII, derived from Archaeoglobus fulgidus, which cleaves a structure formed by the hybridization of two overlapping oligonucleotide probes to a target nucleic acid strand. This method, termed the Invader assay, can discriminate single-base differences. Nine pairs of probes, encompassing five mutations in rpoB and katG that are associated with resistance to either RIF or INH, as well as the corresponding wild-type (drug-susceptible) alleles, were tested using amplified DNA. Fluorescent-labeled cleavage products, ranging from 4 to 13 nucleotides in length, depending on the genotype of the test sample, were separated by denaturing polyacrylamide (20 to 24%) gel electrophoresis and then detected by scanning. All nine alleles could be identified and differentiated on the basis of product size. Multiple mutations at a specific rpoB nucleotide in target PCR products could be identified, as could mutants that were present at > or =0.5% of the total population of target sequences. The Invader assay is a sensitive screen for some mutations associated with antituberculosis drug resistance in amplified gene regions.
Cooksey
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RC
Division of AIDS, STD, and TB Laboratory Research, Atlanta, Georgia 30333, USA. rcc1@cdc.gov
Holloway
B P
BP
Oldenburg
M C
MC
Listenbee
S
S
Miller
C W
CW
D000995:Antitubercular Agents / Q000494:pharmacology*; D001426:Bacterial Proteins; D004252:DNA Mutational Analysis / Q000379:methods*; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D004352:Drug Resistance, Microbial / Q000235:genetics; D005069:Evaluation Studies as Topic; D007538:Isoniazid / Q000494:pharmacology*; D009154:Mutation; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics; D010544:Peroxidases / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D012293:Rifampin / Q000494:pharmacology*
D000995:Antitubercular Agents; D001426:Bacterial Proteins; D010544:Peroxidases; C076136:catalase HPI; D012321:DNA-Directed RNA Polymerases; C105078:RNA polymerase beta subunit; D007538:Isoniazid; D012293:Rifampin