A mutation in the 65,000 Dalton heat shock protein gene, commonly used for molecular identification of non-tuberculous mycobacteria, leads to the misidentification of Mycobacterium malmoense as Mycobacterium marinum.

We describe a case of a Mycobacterium isolated from a patient with cervical lymphadenitis which was initially identified by hsp65 RFLP as Mycobacterium marinum. Sequence analysis of the hsp65 DNA fragment and the 16S rDNA signature sequence, however, led to the identification of Mycobacterium malmoense. A point mutation in one of the restriction sites had shifted the M. malmoense typical RFLP pattern to the M. marinum specific RFLP pattern. As a consequence, care should be taken when identifying mycobacteria with the use of one molecular technique, only.

Species-specific identification of mycobacterial 16S rRNA PCR amplicons using smart probes.

Due to growing problems with new emerging pathogens, cost-effective and manageable methods for their accurate identification in routine diagnostics are urgently required. Of particular importance is the genus Mycobacterium with its more than 100 species. Identification of these species is hampered by their slow growth in the laboratory and by the obligate need for DNA sequence analysis. To provide a fast and reliable diagnostic tool, we developed a novel approach using fluorescently labeled DNA hairpin structures (smart probes) for selective and sensitive detection of mycobacterial 16S rDNA PCR amplicons in homogeneous and heterogeneous assays. Smart probes are singly labeled hairpin-shaped oligonucleotides bearing a fluorescent dye at the 5'-end, which is quenched by guanosine residues in the complementary stem. Upon hybridization to target sequences, a conformational change occurs reflected in an increase in fluorescence intensity. Using optimized parameters for hybridization experiments we established a reliable method for the specific detection of Mycobacterium tuberculosis (M. tuberculosis complex) and Mycobacterium xenopi (member of the atypical mycobacteria) with a detection sensitivity of approximately 2 x 10(-8) M in homogeneous solution. The specificity of the smart probes designed is demonstrated by discrimination of M. tuberculosis and M. xenopi against 15 of the most frequently isolated mycobacterial species in a single assay. In combination with a microsphere-based heterogeneous assay format, the technique opens new avenues for the detection of pathogen-specific DNA sequences with hitherto unsurpassed sensitivity.

Different molecular methods for the identification of rarely isolated non-tuberculous mycobacteria and description of new hsp65 restriction fragment length polymorphism patterns.

Analysis of heat shock protein 65 (hsp65) gene restriction fragment length polymorphism (RFLP) is done frequently to identify non-tuberculous mycobacteria (NTM) on a genetic basis. Here we report the results of analysing the hsp65 patterns of some rarely isolated NTM for which patterns have not been published before (Mycobacterium bohemicum, Mycobacterium hassiacum, Mycobacterium heckeshornense, Mycobacterium monacense, and Mycobacterium triplex). Furthermore new hsp65-variants for Mycobacterium interjectum (type II), Mycobacterium mucogenicum (type V), Mycobacterium gordonae (type VIII) and Mycobacterium paraffinicum (perhaps synonymous to Mycobacterium scrofulaceum) are described. All species were characterised by hsp65-RFLP, sequencing a 441-bp fragment of the hsp65 gene and sequencing the hypervariable region of the 16S rDNA. Additional data for less frequently isolated mycobacteria are provided and the hitherto described data for the Mycobacterium gordonae complex are summarised. Although the hsp65-RFLP analysis turned out to be a useful method a number of restraints (lack of standardisation, slight variability in fragment length) limits its broader use. Reliable identification of NTM needs, however, more than one molecular method. Identification results obtained by applying different methods yielded conflicting results. Confusion may be caused by older data base entries which are not updated and not longer reflect the actual systematic and taxonomy of the genus Mycobacterium.