Genotypic determination of Mycobacterium tuberculosis antibiotic resistance using a novel mutation detection method, the branch migration inhibition M-tuberculosis antibiotic resistance test

A novel method for the detection of any alteration within a defined sequenc e has recently been demonstrated (A. Lishanski, N, Kurn, and E. F. Ullman, Nucleic Acids Res. 28:E42, 2000; A. Lishanski, Clin, Chem, 46:9, 2000), Ess ential to this method are the generation of partial duplexes that are capab le of forming four-stranded structures and the ability to detect inhibition of branch migration in these structures (I. G. Panyutin and P. Hsieh, J. M ol. Biol. 230:413-124, 1993). Inhibition of branch migration indicates the presence of sequence alteration, This mutation detection method, termed bra nch migration inhibition (BMI), is suitable for the detection of drug resis tance in M. tuberculosis, which is frequently associated with multiple muta tions within known genes. We describe the genotypic determination of the ri fampin (RMP) and pyrazinamide (PZA) susceptibilities of M. tuberculosis iso lates, using BMI coupled with the luminescence oxygen channeling immunoassa y (LOCI) (E. F. Ullman et al., Proc. Natl. Acad. Sci. USA 91:5426-5430, 199 4). RMP and PZA resistances are associated with multiple mutations within t he rpoB and pncA genes, respectively, M. tuberculosis genomic DNA samples p repared from 46 clinical isolates were used for genotypic determination of RMP resistance in a "blind study." Similarly, PW resistance was determined using genomic DNA samples prepared from 37 clinical isolates. Full agreemen t of the genotypic and phenotypic determinations of drug susceptibility was demonstrated, RR-IP susceptibility determination directly from cells of 10 clinical isolates grown in culture was also demonstrated. The genotypic re sult of only 1 out of 10 isolates did not agree with the phenotypic suscept ibility testing result, Sequence analysis of the rpoB gene of this clinical isolate revealed a single base substitution, most likely a silent point mu tation. The new BMI-LOCI mutation detection method is a rapid and accurate procedure for the genotypic determination of the RMP and PZA susceptibiliti es of M. tuberculosis clinical isolates, BMI can also be detected by using commercially available automated enzyme-linked immunosorbent assay plate fo rmats (Lishanski et al., Nucleic Acids Res. 28:E42, 2000).