Molecular definition of unique species status of Mycobacterium w; a candidate leprosy vaccine strain.

Mycobacterium w, a candidate leprosy vaccine strain, is an atypical cultivable mycobacterium. Based on its growth and metabolic properties, M. w was listed in Runyon Group IV, along with other rapid growers such as M. fortuitum, M. smegmatis, M. chelonae and M. vaccae. However, M. w was not fully identical to any one of these. In the present study, a molecular biology approach was used to define the species identity of M. w in a manner that allows reliable comparison to be made with over 30 known mycobacterial species. A 383-bp region, present at the amino terminus of the conserved mycobacterial 65-kDa gene, has been polymerase chain reaction (PCR) amplified in M. w and DNA sequence was determined. A comparison of the M. w DNA sequence with those of M. tuberculosis, M. avium, M. paratuberculosis and M. fortuitum revealed a species-specific polymorphism, i.e., the presence of nucleotide substitutions unique to M. w. In an alternate approach, a 441-bp region, also a part of the 65-kDa gene, has been PCR amplified in M. w and a Hae III restriction pattern was generated.(ABSTRACT TRUNCATED AT 250 WORDS)

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 lambda gt11 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.

Repetitive DNA sequence of Mycobacterium tuberculosis: analysis of differential hybridization pattern with other mycobacteria.

In order to generate specific DNA probes for Mycobacterium tuberculosis, restriction fragment length analysis was carried out with some of the mycobacterial species that fall within the tuberculosis complex. The presence of specific bands of 5.6 kb and 4.8 kb was revealed in the AluI DNA digest of M. tuberculosis. The hybridization profile of the 5.6-kb AluI DNA sequence, as judged by the Southern blot and dot blot hybridization experiments, revealed the presence of this sequence in M. tuberculosis H37Rv, M. tuberculosis H37Ra, M. bovis BCG as multiple copy and M. kansasii as single copy but this sequence was not present in M. avium, M. smegmatis, or M. vaccae genomes. Genomic clones corresponding to the 5.6-kb AluI fragment from M. tuberculosis H37Rv library made in the lambda gt11 expression vector were isolated.