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Characterization of Mycobacterium tuberculosis complex direct repeat sequence for use in cycling probe reaction

Cycling probe technology (CPT) is a unique and simple method for the detection of specific target sequences. CPT utilizes a chimeric DNA-RNA-DNA probe providing an RNase H-sensitive scissile linkage when bound to a complementary target sequence. For this study a diagnostic assay based on CPT was developed for the detection of the 36-bp direct repeat (DR) region in Mycobacterium tuberculosis. To determine the feasibility of using the DR for detecting M. tuberculosis by CPT, a wide variety of mycobacteria were tested by Southern blot hybridization with three DR probes to verify their specificity. The entire DR region of Mycobacterium bovis 401 was sequenced, and the data were used to design a PCR assay that would allow us to estimate the number of DRs present in a variety of strains. A CPT assay which uses a probe complementary to the DR region was developed and evaluated with synthetic targets and genomic DNA from mycobacteria. In summary, the 36-bp DR provides an attractive target for detecting M. tuberculosis because the sequence is present in high copy numbers in the genome, is specific for the M. tuberculosis complex, and is found in strains that lack IS6110.