Novel polymorphic region of the rpoB gene containing Mycobacterium species-specific sequences and its use in identification of mycobacteria

Abstract

Sequence analysis of a specific region of the mycobacterium rpoB gene in 35 mycobacterial strains representing 26 different mycobacterial species of clinical importance showed that there exists a highly polymorphic region. Based on the sequences of the polymorphic region, the oligonucleotide probes of 14 mycobacterial species with relatively high clinical importance were designed and shown to be specific to their corresponding mycobacterial species by dot blot hybridization. The results showed that the probes designed in this study are highly specific to each mycobacterial species, which suggests that these sequences may be useful for the species identification of mycobacteria.

FIG. 1.

Multialignment sequences identified in the rpoB region of 35 strains of 26 mycobacterial species of clinical importance. The software used for the alignment of multiple sequences was obtained from reference . Sequences with high consensus are shown as red letters, low consensus as blue letters, and neutral consensus as black letters. Among the 360-bp sequences identified in this study, only the 216-bp region of the upstream sequences is shown, since the downstream sequences have been reported elsewhere. The sequences shown here have been submitted to GenBank.

FIG. 1.

Multialignment sequences identified in the rpoB region of 35 strains of 26 mycobacterial species of clinical importance. The software used for the alignment of multiple sequences was obtained from reference . Sequences with high consensus are shown as red letters, low consensus as blue letters, and neutral consensus as black letters. Among the 360-bp sequences identified in this study, only the 216-bp region of the upstream sequences is shown, since the downstream sequences have been reported elsewhere. The sequences shown here have been submitted to GenBank.

FIG. 2.

The results of the dot blot hybridizations using each mycobacterial species-specific oligomer probe derived from the novel region of the rpoB genes of species. Dot blot hybridizations were conducted using probes specific for M. tuberculosis complex and M. gastri. The PCR-amplified products from 48 mycobacterial species were blotted on the membrane, and this was followed by hybridization with probes. The identification numbers in the membranes are matched with numbers and species names in Table 1.

FIG. 3.

Dot blot hybridizations using the M. avium specific oligomer probe. The membrane contained PCR products amplified from 36 clinical isolates of mycobacteria, including six isolates of M. avium species. The clinical isolates were identified by conventional culture and biochemical tests at the KIT.