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

Abstract

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.

Mapping of IS 6110 Insertion Sites in Two Epidemic Strains of Mycobacterium tuberculosis

“…The first five spacers between the DRs on the left side of IS6110 are not present in H37Rv. Four are identical to those described for M. bovis strain 401, and one is a unique spacer recently reported (2,7,12,17,25). The sequence from spacer 51 to the region of MTCY16B7 outside the last DR is identical to that in H37Rv.…”

“…2). In contrast to H37Rv, Mycobacterium bovis BCG, and M. bovis 401, this copy of IS6110 has been inserted in a different site within a copy of the DR (2,7,12,17). The DR sequence interrupted by IS6110 in the above strains is flanked by three C's.…”

New Rapid Polymerase Chain Reaction‐Immunochromatographic Assay for Porphyromonas gingivalis

“…Recently, several unique methods to detect specific target substrates in Mycobacterium tuberculosis 19,20 and Staphylococcus aureus 21 instead of bacterial culture methods have been reported. Anaerobic bacteria could become nonviable after sampling, during transport, and analysis; thus, a technique such as culturing would underestimate the number of bacteria in periodontal pockets.…”

Evaluation of a Urine-Based Rapid Molecular Diagnostic Test with Potential to Be Used at Point-of-Care for Pulmonary Tuberculosis

“…Forward and reverse primers targeting the MTB-specific direct repeat (DR) region were designed. 26 These primers bound to a 38-bp sequence present in the 4392-bp DR region. 27 The 5 0 -Cy5elabeled forward primer had a length of 17 bp.…”