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

Abstract

We evaluated the usefulness of PCR assays that target the internal transcribed spacer (ITS) region for identifying mycobacteria at the species level. The conservative and species-specific ITS sequences of 33 species of mycobacteria were analyzed in a multialignment analysis. One pair of panmycobacterial primers and seven pairs of mycobacterial species-specific primers were designed. All PCRs were performed under the same conditions. The specificities of the primers were tested with type strains of 20 mycobacterial species from the American Type Culture Collection; 205 clinical isolates of mycobacteria, including 118 Mycobacterium tuberculosis isolates and 87 isolates of nontuberculous mycobacteria from 10 species; and 76 clinical isolates of 28 nonmycobacterial pathogenic bacterial species. PCR with the panmycobacterial primers amplified fragments of approximately 270 to 400 bp in all mycobacteria. PCR with the M. tuberculosis complex-specific primers amplified an approximately 120-bp fragment only for the M. tuberculosis complex. Multiplex PCR with the panmycobacterial primers and the M. tuberculosis complex-specific primers amplified two fragments that were specific for all mycobacteria and the M. tuberculosis complex, respectively. PCR with M. avium complex-, M. fortuitum-, M. chelonae-, M. gordonae-, M. scrofulaceum-, and M. szulgai-specific primers amplified specific fragments only for the respective target organisms. These novel primers can be used to detect and identify mycobacteria simultaneously under the same PCR conditions. Furthermore, this protocol facilitates early and accurate diagnosis of mycobacteriosis.

FIG. 1

Alignment of the mycobacterial ITS sequences. The alignment includes conserved and polymorphic regions derived from the mycobacterial species. The conserved sequences are in bold print, and the polymorphic sequences are in italic print. Dashes represent deletions, and asterisks represent identity.

FIG. 2

Genus-specific amplification of mycobacterial ITS by primers ITS-F and mycom-2. Lanes M, 100-bp size markers; lanes C, negative control; lane 1, M. abscessus ATCC 19977; lane 2, M. agri ATCC 27406; lane 3, M. asiaticum ATCC 25276; lane 4, M. austroafricanum ATCC 33464; lane 5, M. avium ATCC 25291; lane 6, M. bovis ATCC 19210; lane 7, M. chelonae ATCC 35752; lane 8, M. flavescens ATCC 14474; lane 9, M. fortuitum ATCC 6841; lane 10, M. gordonae ATCC 14470; lane 11, M. intracellulare ATCC 13950; lane 12, M. kansasii ATCC 12478; lane 13, M. phlei ATCC 354; lane 14, M. scrofulaceum ATCC 19981; lane 15, M. smegmatis ATCC 21701; lane 16, M. szulgai ATCC 35799; lane 17, M. terrae ATCC 15755; lane 18, M. triviale ATCC 23292; lane 19, M. tuberculosis H37Rv; lane 20, M. vaccae ATCC 15483.

FIG. 3

PCR with each pair of mycobacterial species-specific primers. From top to bottom, primers TBF and TBR, MACF and MACR, FORF and FORR, CHEF and CHER, GORF and GORR, SZUF and SZUR, and SCOF and SCOR. Lanes M, 100-bp DNA ladder size markers; lane C, negative control; lane 1, M. tuberculosis H37Rv; lane 2, M. bovis; lane 3, M. avium; lane 4, M. intracellulare; lane 5, M. fortuitum; lane 6, M. chelonae; lane 7, M. gordonae; lane 8, M. szulgai; lane 9, M. terrae; lane 10, M. scrofulaceum.

FIG. 4

Multiplex PCR with panmycobacterial and M. tuberculosis-specific primers. Lanes M, 100-bp DNA ladder size markers; lane C, negative control; lane 1, M. tuberculosis H37Rv; lane 2, M. bovis; lane 3, M. avium; lane 4, M. intracellulare; lane 5, M. fortuitum; lane 6, M. chelonae; lane 7, M. gordonae; lane 8, M. szulgai: lane 9, M. terrae; lane 10, M. scrofulaceum.