Discrimination of Mycobacterium tuberculosis strains by PCR

Abed et al. (1) reported a PCR-based method to discriminate among Mycobacterium tuberculosis strains. They first amplified the 16S-23S ribosomal DNA (rDNA) internal transcribed spacer using specific primers. They then reamplified using a single arbitrary primer, resulting in products of variable sizes. They concluded that ‘‘a high level of discriminatory power was observed with this method because of the high degree of polymorphism characteristic of the 16S-23S spacer region. ’’ Most bacteria have multiple rRNA operons and thus multiple copies of the 16S-23S rDNA internal transcribed spacer (ITS). However, slowly growing mycobacteria are unusual in having a single rRNA operon (2). The single-copy ITS has been sequenced in more than 80 mycobacterial strains, including more than 20 strains of theM. tuberculosis complex. Strains of some mycobacterial species vary in their ITS sequences, but all strains of the M. tuberculosis complex have identical ITS sequences (4, 5). The variably sized PCR products reported by Abed et al. (1) did not result from sequence polymorphisms in the ITS. Presumably, they represented arbitrary priming in other parts of the chromosome. Rapid M. tuberculosis strain differentiation has important public health and therapeutic implications. Methods using arbitrary primer and specific-primer PCR have been reported (3, 6, 8, 9). Arbitrary primer PCR (AP-PCR [10], also referred to as random amplified polymorphic DNA [RAPD]) relies on low-stringency primer annealing. Patterns produced by APPCR vary greatly with minor changes in amplification conditions, and it is difficult to achieve reproducible profiles (8). Also, M. tuberculosis strains have highly conserved DNA sequences in a variety of genes and intergenic regions (4, 5, 7). Since the underlying DNA sequences are highly conserved, most AP-PCR products are also conserved (8). I have attempted to differentiate M. tuberculosis strains using AP-PCR with a variety of primers. In my hands, the method was either nondiscriminative or nonreproducible. Abed et al. presented no data on the reproducibility of the variable profiles they report (1). In contrast, specific-primer PCR uses primers complementary to a particular chromosomal segment. Targets for M. tuberculosis strain differentiation have included tandem DNA repeats, drug resistance genes, or mobile insertion elements (3, 6, 9). Polymorphisms have been identified in the PCR products by length, restriction digestion, sequencing, or single-strand conformation. Specific-primer PCR is reproducible (3, 6, 9) and applicable to clinical specimens (9), but most of the described methods have had limited discriminative power. Future reports of AP-PCR strain differentiation should include detailed evidence of reproducibility. The same strain should be analyzed repeatedly on different days. Analysis should include isolates of reference strains from multiple sources, cultures grown on different media, cultures of different ages, and DNA isolated by different methods (8). Reports of specific-primer PCR should include some evidence that the targeted region was actually amplified (e.g., results of direct sequencing [3, 9] or Southern hybridization [6]), to exclude the possibility that polymorphisms are due to arbitrary priming. The ability of any method to discriminate different strains and to group epidemiologically related strains should be evaluated by using a well-characterized strain collection (3, 6, 9). The collection should ideally be coded to minimize bias in the interpretation of results.