Differentiation between Mycobacterium tuberculosis and Mycobacterium avium by amplification of the 16S-23S ribosomal DNA spacer

Abstract

Differentiation between Mycobacterium tuberculosis and M. avium is helpful for the treatment of disseminated mycobacterial infection in AIDS patients. This can traditionally be done by time-consuming biochemical tests or with Accuprobe. Previously, PCR restriction enzyme analysis (PCR-REA) of the 16S-23S rRNA gene spacer was shown to be able to identify a limited number of strains of Mycobacterium. In this study the method was improved by using more specific primers and was tested with 50 clinical isolates of M. tuberculosis and 65 clinical isolates of M. avium complex. Probes specific to the spacers of M. tuberculosis and M. avium were also tested. Both M. tuberculosis and M. avium could be reliably identified either by PCR-REA or by PCR-hybridization, with the results completely agreeing with those obtained by biochemical tests and with the Accuprobe, respectively. The method may therefore be useful as an alternative in-house method for identification of the bacteria.

FIG. 1

Dendrogram for differentiating Mycobacterium species by amplified 16S-23S rRNA gene length, with 16SC and 23SG as primers, and REA of the amplified products. The numbers of isolates are indicated in parentheses.

FIG. 2

HaeIII-digested PCR products of some of the members of MAC. The markers on both sides are plasmid pBR322 digested with MspI. The marker in the middle is the 100-bp ladder. Lanes 1 to 10, M. avium; lanes 11 to 13, M. intracellulare; lane 14, unclassified MAC. The species of the samples in the other three lanes are labeled.

FIG. 3

Dot blot hybridization of the amplified products of some isolates belonging to the M. avium complex. A1, amplified product of M. tuberculosis H37Rv; C4, C5, and D4, amplified products of unclassified MAC; E3 to E5, amplified products of M. intracellulare.