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

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Abstract
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.