MOLECULAR-CLONING AND CHARACTERIZATION OF CONTIGUOUSLY LOCATED REPETITIVE AND SINGLE-COPY DNA-SEQUENCES OF MYCOBACTERIUM-TUBERCULOSIS - DEVELOPMENT OF PCR-BASED DIAGNOSTIC ASSAY

Screening of a lambdagt11 genomic library has been used as an approach for molecular cloning of the Mycobacterium tuberculosis repetitive DNA shown to be present on a previously described 5.6-kb Alu I genomic fragment. The recombinant clone R18.8.2, which strongly hybridized with the radiolabeled 5.7-kb Alu fragment, carried two Eco RI inserts of 2 kb and 1.4 kb in size. Southern hybridization of each of these fragments to restriction endonuclease-cleaved M. tuberculosis DNA clearly demonstrated the 2 kb to contain the repetitive DNA sequence, while the 1.4 kb is represented in a single copy. When replica plaque lifts from the genomic library were probed, the 5.6-kb genomic fragment and the cloned 2-kb repetitive insert hybridized to an identical number of plaques, indicating the similarity and the high copy number of the repeating unit shared by the two fragments. Restriction mapping and Southern hybridization patterns indicated that the 2-kb repetitive and the 1.4-kb single-copy DNA sequences originated from a contiguous piece of genomic DNA. Both fragments were found to be unique to members of the M. tuberculosis complex, except that the 2-kb insert exhibited a weak hybridization with M. kansasii DNA. Finally, a 169-bp region from one end of the single-copy sequence has been amplified by polymerase chain reaction (PCR) in a manner specific to members of the M. tuberculosis complex. The sensitivity of the PCR was such that as few as 9-10 bacilli could be detected.