Single nucleotide polymorphic (SNP) typing

SNP typing was then performed on these samples using lineage 3 and 4 markers as earlier described in Micheni et al.⁴⁴ to screen for the commonly occurring MTBC lineages in this region while SNP typing was performed as previously described⁴⁴ using lineage 3 and 4 specific primers (Rv004C for MTB L4-U, Rv2962C for MTB L4-NU and Rv0129C for MTB L3) and their accompanying hybridization probes. Briefly, the assays were performed in 20 µl reaction mixture containing 3.75 μl of PCR water, 1.25 μl (0.5 μM final concentration) of each primer, 0.625 μl (0.25 μM final concentration) of each probe, 9.5 μl of 2X Lunar^® Universal genotyping master mix, and 3 μl (5–50 ng) of extracted genomic DNA. RT-PCR was carried out in a Bio-Rad CFX96 Touch™ that was programmed for PCR amplification and a melting curve stage. For each of the three uniplex assays, the amplification stage consisted of a pre-PCR stage performed at 95 °C for 10 min, an amplification stage with denaturation at 95 °C for 30 s, primer annealing (50 °C for Rv004C or 52 °C for Rv0129C or 51 °C for Rv2962C) for 30 s and extension at 60 °C for 30 s for 45 cycles. The melting curve analysis consisted of denaturation of the amplicons at 95 °C for 10 s to produce single-stranded DNA, probe annealing temperature at 65 °C for 05 s with a continuous acquisition mode to allow capture the fluorescence and probe melting temperature ranging from 40–80 °C. The MTBC lineages were identified based on differences in melting temperature (Tm). H37Rv (L4-NU), kc32969 (L4-U) and delicus (L3) genomic DNA were used as positive control while non-template mix as a negative control.