Reverse dot blot assay (insertion site typing) for precise detection of sites of IS6110 insertion in the Mycobacterium tuberculosis genome

We have developed an amplification-based reverse dot blot assay for the det ection of specific sites of insertion of the Mycobacterium tuberculosis ins ertion sequence IS6110, In this assay, a set of biotin-labeled amplicons re presenting the various copies of IS6110 and their flanking sequences is gen erated by linker-mediated PCR, The amplicons are then hybridized to immobil ized oligonucleotide probes that are specific for known IS6110 insertion si tes, The method was evaluated using an array of oligonucleotide probes corr esponding to IS6110 insertion sites from M, tuberculosis strains CDC1551, E rdman, and H37Rv, and multidrug-resistant strain W. A set of 72 DNA samples from 60 M. tuberculosis clinical isolates was analyzed for the presence or absence of these insertion sites, and the assay was found to be highly rep roducible. This method of identifying insertion sites has been named "insit e" and can be used for the genotyping of M. tuberculosis complex strains ba sed on IS6110 insertion site profiles.