Detection and identification of mycobacteria by amplification of the internal transcribed spacer regions with genus- and species-specific PCR primers

We evaluated the usefulness of PCR assays that target the internal transcri bed spacer (ITS) region for identifying mycobacteria at the species level. The conservative and species-specific ITS sequences of 33 species of mycoba cteria were analyzed in a multialignment analysis. One pair of panmycobacte rial primers and seven pairs of mycobacterial species-specific primers were designed. All PCRs were performed under the same conditions. The specifici ties of the primers were tested with type strains of 20 mycobacterial speci es from the American Type Culture Collection; 205 clinical isolates of myco bacteria, including 118 Mycobacterium tuberculosis isolates and 87 isolates of nontuberculous mycobacteria from 10 species; and 76 clinical isolates o f 28 nonmycobacterial pathogenic bacterial species. PCR with the panmycobac terial primers amplified fragments of approximately 270 to 400 bp in all my cobacteria. PCR with the M. tuberculosis complex-specific primers amplified an approximately 120-bp fragment only for the M. tuberculosis complex. Mul tiplex PCR with the panmycobacterial primers and the M. tuberculosis comple x-specific primers amplified two fragments that were specific for all mycob acteria and the M. tuberculosis complex, respectively, PCR with M. avium co mplex-, M. fortuitum-, M. chelonae-, M. gordonae-, M. scrofulaceum-, and M. szulgai-specific primers amplified specific fragments only for the respect ive target organisms. These novel primers can be used to detect and identif y mycobacteria simultaneously under the same PCR conditions. Furthermore, t his protocol facilitates early and accurate diagnosis of mycobacteriosis.