DNA amplification and reverse dot blot hybridization for detection and identification of mycobacteria to the species level in the clinical laboratory

Abstract

A method incorporating DNA amplification and reverse dot blot hybridization for the detection and identification of mycobacteria to the species level is described. The amplification procedure allowed for the incorporation of digoxigenin-labeled UTP, which was detected by chemiluminescence, removing the need for radioactivity. Using a set of primers and probes from the gene for the 65-kDa heat shock protein of mycobacteria, previously reported in the literature, the reverse dot blot method correctly identified 12 of the 12 M. tuberculosis isolates and 45 of the 50 M. avium complex isolates. Two of the nonhybridizing M. avium complex isolates were reidentified as M. xenopi. The other three nonhybridizing M. avium complex isolates, which were identified as M. intracellulare, hybridized with the probe for M. tuberculosis, as did two ATCC strains of M. intracellulare. The amplified DNA of M. intracellulare was sequenced, and the sequence was compared with the sequence from M. tuberculosis. The sequence for M. avium differed from M. tuberculosis by 5 of 20 bases. The sequence for M. intracellulare differed from M. tuberculosis by 2 of 20 bases, but this difference did not result in sufficient thermal instability to affect hybridization. The use of chemiluminescence allowed as few as 10(2) CFU to be detected. The format of the assay is readily applicable for implementation in the clinical laboratory.