Ladder 100 bp plus opti dna marker

A multiplex PCR developed for the detection of the RD^Rio pattern was performed using a two set of primers (Supplementary Figure S1): (1) a set of primers that target the IS1561’ fragment, located inside the region between the genes Rv3346c and Rv3355c, and used as a marker for wild type M. tuberculosis (530 bp); (2) another set of primers is used to flank the RD^Rio locus and bridge the deletion in RD^Rio strains (1175 bp) (36 (link)). The PCR reaction was prepared as follows: 12.5 μL of GoTaq^® Green Mastermix (Promega, Wisconsin, United States) (1X), 0.5 μL of each primer (0.2 μM), 5 μL of mycobacterial DNA and completed with Nuclease-Free water to a final volume of 25 μL. The multiplex PCR program was established as follows: initial denaturation at 94°C for 3 min, 35 cycles at 94°C for 30 s, 60°C for 30 s, 72°C for 1 min 30 s, and a final extension step of 72°C for 10 min. PCR products were run in 1.5%UltraPure^™ Agarose (Invitrogen, California, United States) gels of 15 cm × 10 cm in 0.5X Tris-boric acid-EDTA (TBE) buffer at 100 V for 2 h using a ladder 100 bp Plus Opti-DNA Marker (Cat. No.: G016, Applied Biological Materials Inc., British Columbia, Canada) for size determination (22 (link), 36 (link)).

The method is PCR-based and allows the detection of different mycobacterial interspersed repetitive units (MIRU) located at multiple loci in the MTBC genome. Each MIRU allele is identified by a number, thus generating a numerical profile that is used for genotyping studies (5 (link), 31 (link), 32 (link)). Amplicons were observed in 2% UltraPure™ Agarose (Invitrogen, California, USA) gels of 15 cm x 10 cm in 0.5X Tris-boric acid-EDTA (TBE) buffer at 100 V for 3 h using a ladder 100-bp Plus Opti-DNA Marker (Cat.No.: G016, Applied Biological Materials Inc., British Columbia, Canada) for size determination. MIRU allele identification was performed according to Supply et al. (33 ).