Can random amplified polymorphic DNA analysis of the 16S-23S spacer region of Mycobacterium tuberculosis differentiate between isolates?

In response to the article by Abed et al. (1) describing the discrimination of Mycobacterium tuberculosis isolates by 16S23S spacer region-based random amplified polymorphic DNA (RAPD) analysis, we have the following comments. We have previously reported a PCR-based strategy for accessing the 16S-23S intergenic region of eubacteria (2) and subsequently generated the DNA sequence of this region from 13 strains of the M. tuberculosis complex (M. tuberculosis, M. bovis, M. microti) (7). The DNA sequences of the 16S-23S region of these three species were identical. In a separate study, the DNA sequences of the 16S-23S intergenic region from an additional 13 strains of the M. tuberculosis complex were generated (4) and found to be identical to the sequence data from our study. These also showed extensive DNA sequence homology throughout the M. tuberculosis complex in this region. Other workers have also analyzed the DNA sequence from this region of these species and have had similar results (9, 10, 12). The rate of base substitution between isolates of a species in this region is less than 1% (8). On the basis of these data, the PCR primers described by Abed et al. (1) would be expected to amplify a 364-bp fragment from the M. tuberculosis complex. The generation of four bands (526, 471, 333, and 303 bp) from three M. tuberculosis strains by using the universal PCR primers shown in Fig. 1 is therefore surprising. The absence of the anticipated size product as indicated by the DNA sequence of this region of M. tuberculosis would suggest that nonspecific amplification has occurred. Additionally, universal PCR primers are capable of amplifying any bacterial target that may have been in the reaction cocktail (including DNA from Thermus aquaticus or Escherichia coli, if the Taq polymerase were produced from a cloned fragment expressed in that organism). Because of the absence of a template-free negative control from this study, this possibility cannot be eliminated. The fact that tRNA genes have not been identified in the 16S-23S spacer region of slowly growing mycobacteria (5, 6, 11, 12) combined with the fact that there is just one rRNA operon per genome (3, 10 –12) makes it unlikely that the four bands generated are all genuine amplicons from the 16S-23S intergenic region of M. tuberculosis. The RAPD patterns shown in Fig. 3 of this study could be due to the priming events of the RAPD primer combined with the actions of the initial two primers on the M. tuberculosis genomic DNA which were carried through by the ethanol precipitation of the amplicons from the primary amplification. Equally, it may be that amplification of another region ofM. tuberculosis is occurring under the PCR conditions used in the primary amplification, which is capable of generating amplicons that are subsequently useful to differentiate between isolates under the RAPD conditions described (i.e., universal primers and M. tuberculosis genomic DNA also in the RAPD reactions). However, the presence of these additional primers, genomic DNA, and amplicons of possible unknown origins in the RAPD analysis suggests that further studies are required to prove the reliability and source of this M. tuberculosis differentiation method.