Real-time PCR for single-nucleotide polymorphism detection in the 16S rRNA gene as an indicator for extensive drug resistance in Mycobacterium tuberculosis

Abstract

A real-time PCR screening system was established for rapid detection of single-nucleotide polymorphisms (SNPs) at positions 1401, 1402 and 1484 of the 16S rRNA gene of Mycobacterium tuberculosis leading to resistance to amikacin, kanamycin and capreomycin. Resistances to the respective drugs may indicate the presence of an extensively drug-resistant (XDR) strain of M. tuberculosis. Fifty-seven M. tuberculosis isolates that tested phenotypically susceptible or resistant to amikacin, capreomycin or both were subjected to 1401-2/1484 real-time PCR to screen for SNPs in the respective rrs region. 1401-2 and 1484 wild-type and mutant M. tuberculosis strains displayed distinct melting peaks. Of the cross-resistant strains, 86.7% displayed A1401G SNPs, 76.9% of amikacin-resistant strains did not display rrs SNPs and one capreomycin-resistant strain showed a C1402T SNP. Phenotypic drug susceptibility testing takes several weeks, but with the 1401-2/1484 real-time PCR a preliminary diagnosis can be made within a few hours. SNPs in the rrs region are not exclusively involved in the development of resistances to amikacin and capreomycin. However, 80.0% of XDR-tuberculosis samples tested were detected with the real-time PCR screening assay of the present study.