Species-Specific Identification of Mycobacterial\n16S rRNA PCR Amplicons Using Smart Probes

Abstract

Due to growing problems with new emerging pathogens,\ncost-effective and manageable methods for their accurate\nidentification in routine diagnostics are urgently required.\nOf particular importance is the genus <i>Mycobacterium</i>\nwith its more than 100 species. Identification of these\nspecies is hampered by their slow growth in the laboratory\nand by the obligate need for DNA sequence analysis. To\nprovide a fast and reliable diagnostic tool, we developed\na novel approach using fluorescently labeled DNA hairpin\nstructures (smart probes) for selective and sensitive\ndetection of mycobacterial 16S rDNA PCR amplicons in\nhomogeneous and heterogeneous assays. Smart probes\nare singly labeled hairpin-shaped oligonucleotides bearing\na fluorescent dye at the 5‘-end, which is quenched by\nguanosine residues in the complementary stem. Upon\nhybridization to target sequences, a conformational change\noccurs reflected in an increase in fluorescence intensity.\nUsing optimized parameters for hybridization experiments\nwe established a reliable method for the specific detection\nof <i>Mycobacterium tuberculosis (M. tuberculosis </i>complex<i>)</i> and <i>Mycobacterium</i> <i>xenopi</i> (member of the atypical mycobacteria) with a detection sensitivity of ∼2 × 10^-8\nM in homogeneous solution. The specificity of the smart\nprobes designed is demonstrated by discrimination of <i>M.\ntuberculosis</i> and <i>M. xenopi</i> against 15 of the most\nfrequently isolated mycobacterial species in a single assay.\nIn combination with a microsphere-based heterogeneous\nassay format, the technique opens new avenues for the\ndetection of pathogen-specific DNA sequences with hitherto unsurpassed sensitivity.