Rapid detection and species identification of Mycobacterium spp. using real-time PCR and DNA-microarray.

Infections with mycobacteria are an important issue in public health care. Here we present a "proof-of-principle" concept for the identification of 37 different Mycobacterium species using 5' exonuclease real-time PCR and DNA microarray based on the region upstream of the 65 kDa heat shock protein. With our two PCR probes, one complementary to all mycobacteria species, the other specific for the M. tbc-complex, 34 species were properly classified by real-time PCR. After reamplification and hybridization to a DNA microarray, all species showed a specific pattern. All 10 blindly tested positive cultures revealed a positive real-time PCR signal with the genus probe. After reamplification and hybridization, six samples could unambiguously be identified. One sample showed a mixture of presumably three species-specific patterns and sequencing the 16S rRNA confirmed the presence of a mixture. The hybridization results of three specimens could not be interpreted because the signal to background ratio was not sufficient. Two samples considered as negative controls (LAL Reagent Water (Cambrex) and DNA of Candida albicans) gave neither a genus nor a M. tbc-complex positive PCR signal. Based on these results we consider our method to be a promising tool for the rapid identification of different mycobacteria species, with the advantage of possible identification of mixed infections or contaminations.