Microheterogeneity within rRNA of Mycobacterium gordonae

Walton and Valesco (5) recently described the identification of Myobacterium gordonae from culture by the GenProbe Rapid Diagnostic System (Gen-Probe, Inc., San Diego, Calif.), in which a '25I-labeled DNA probe complementary to M. gordonae rRNA is employed. Although they reported a sensitivity of 98.7% and a specificity of 98.4% for the assay (with 2 consistent negative M. gordonae isolates of 159 total), they also noted certain problems with respect to the hybridization incubation temperature, the density of the culture suspension, and proper function of the sonicator. In particular, fluctuations within the hybridization temperature represented a potential technical pitfall, since raising the temperature of hybridization by as little as 2°C above the optimum of 71 + 1°C reduced the percent hybridization to below the 10% cutoff value. The authors concluded that "in laboratories where the conventional biochemical tests are replaced with the Gen-Probe M. gordonae probe, workers who experience negative hybridization values may inadvertently report false-negative results based on probe results alone." In our laboratory we use direct sequence determination of 16S ribosomal DNA which has been previously amplified by the polymerase chain reaction for fast and accurate identification of mycobacteria from culture (3). We have found that in contrast to other mycobacteria, which show a conservation of the rRNA sequence at the species level (3; unpublished data), M. gordonae exhibits ribosomal DNA variation. As shown in Fig. 1, the rRNA sequence variability of M. gordonae is within a region which, because of its interspecies sequence variability, is a common target for diagnostic species-specific DNA probes (1, 2). Since the thermal stability of rRNA-DNA hybrids is dependent on a proper homology of complementary target and probe sequences, we wonder whether this genetic microheterogeneity of M. gordonae might be responsible for some of the problems encountered by Walton and Valesco.