Evaluation of the invader assay, a linear signal amplification method, foridentification of mutations associated with resistance to rifampin and isoniazid in Mycobacterium tuberculosis

We evaluated a recently described linear signal amplification method for se nsitivity and specificity in detecting mutations associated with resistance to rifampin (RIF) and isoniazid (INH) in Mycobacterium tuberculosis. The a ssay utilizes the thermostable flap endonuclease Cleavase VIII, derived fro m Archaeoglobus fulgidus, which cleaves a structure formed by the hybridiza tion of two overlapping oligonucleotide probes to a target nucleic acid str and. This method, termed the Invader assay, can discriminate single-base di fferences. Nine pairs of probes, encompassing five mutations in rpoB and ka tG that are associated with resistance to Either RIF or INH, as well as the corresponding wild-type (drug-susceptible) alleles, were tested using ampl ified DNA. Fluorescent-labeled cleavage products, ranging from 4 to 13 nucl eotides in length, depending on the genotqpe of the test sample, were separ ated by denaturing polyacrylamide (20 to 24%) gel electrophoresis and then detected by scanning. All nine alleles could be identified and differentiat ed on the basis of product size. Multiple mutations at a specific rpoB nucl eotide in target PCR products could be identified, as could mutants that we re present at greater than or equal to 0.5% of the total population of targ et sequences. The Invader assay is a sensitive screen for some mutations as sociated with antituberculosis drug resistance in amplified gene regions.