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Antimicrobial Agents and Chemotherapy, May 2000, p. 1296-1301, Vol. 44, No. 5
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Evaluation of the Invader Assay, a Linear Signal Amplification Method, for Identification of Mutations Associated with Resistance to Rifampin and Isoniazid in Mycobacterium tuberculosis

Robert C. Cooksey,1,* Brian P. Holloway,2 Mary C. Oldenburg,3 Sonja Listenbee,3 and Carolyn W. Miller3

Division of AIDS, STD, and TB Laboratory Research,1 and Scientific Resources Program,2 National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333, and Third Wave Technologies, Inc., Madison, Wisconsin 537193

Received 20 August 1999/Returned for modification 21 December 1999/Accepted 10 February 2000

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 >= 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.

* Corresponding author. Mailing address: Tuberculosis/Mycobacteriology Branch, Centers for Disease Control and Prevention, Mail stop F-08, Atlanta, GA 30333. Phone: (404) 639-1280. Fax: (404) 639-1287. E-mail: rcc1{at}cdc.gov.

Antimicrobial Agents and Chemotherapy, May 2000, p. 1296-1301, Vol. 44, No. 5
0066-4804/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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