Genotyping based on thermal denaturation of amplification products identifies species of the Mycobacterium tuberculosis complex

摘要

Purpose. To develop a fast and inexpensive genotyping assay to identify the Mycobacterium tuberculosis complex (MTC) species most prevalent in human tuberculosis (TB), based on the thermal denaturation profiles of PCR products from mycobacterial 16S rDNA and three MTC genomic regions of difference (RD).Methodology. Genotypes were determined by the presence and thermal denaturation profiles of the amplicons generated in the ‘preliminary’ PCR mixture (16S rDNA), followed by those of the simultaneous D1 (RD9+, RD1?) and D2 (RD4+, RD4?) PCR mixtures. The 16S rDNA profile identifies the genus Mycobacterium; the absence of any additional RD profile identifies Mycobacterium non-tuberculous (MNT) strains; additional RD4+ and RD9+ profiles without RD1? identify M. tuberculosis; an additional RD4+ profile?per se identifies M. africanum; an additional RD4? profile per se identifies Mycobaterium bovis; additional RD1? and RD4? profiles identify M. bovis BCG.Results. Genotypes of a panel with 44 mycobacterial strains coincided in 16?MB and five non-MTC strains; in the remaining 23 MTC strains, 17 MTB and five MA concordant genotypes and one discordant MB genotype were resolved. The genotypes of 13 human and bovine MTC isolates coincided in all four MB and eight of the nine MTB isolates.Conclusion. Sensitivity, specificity and positive and negative predictive values of the method are 100?% for the genus Mycobacterium, which resolves MB, MTB and MA genotypes. Species/genotype agreement is 97.7?% for the panel and 92.3?% for the MTC isolates. This method may be advantageously used to identify the most prevalent MTC species in humans.