Custom master mix

Spoligotyping was performed at the Aklilu Lemma Institute of Pathobiology, Addis Ababa University. One hundred and seventy‐seven (177) isolates confirmed as M. tuberculosis using para nitro benzoic acid inhibition test, and RD9 typing was further analyzed by spoligotyping according to the standardized protocol (Kamerbeek et al., 1997) and following manufacturer's instructions (reagents from Ocimum Biosolution, custom Master Mix from ABgene). Two (2) isolates DNA were not amplified and therefore not spoligotyped. The presence of spacers was visualized on film as black squares after incubation with streptavidin‐peroxidase and enhanced chemiluminescence detection reagents (RPN 2105 Amersham, GE Healthcare Bio‐Sciences). The spacer hybridization was read by two independent readers; in case of discrepancy, the opinion of a third reader was considered. The patterns were translated into binary and octal format as previously described (Dale et al., 2001). The 43‐digit binary code was converted to 15‐digit octal code (base 8, having the digits 0–7).

All M. tuberculosis complex strains were assayed by spoligotyping using standard protocols [10 (link)] and following manufacturer’s instructions (reagents from Ocimum Biosolution, custom MasterMix from ABgene). The presence of spacers was visualized on film as black squares after incubation with streptavidin-peroxidase and ECL chemiluminescence detection reagents (RPN 2105 Amersham, GE Healthcare Bio-sciences). The spacer hybridization patterns were translated into binary and octal format as previously described [17 (link)]. The 43-digit binary code was converted to 15-digit octal code (base 8, having the digits 0–7) [17 (link)].
The binary codes of the isolates were entered into the SITVIT2 database of the Pasteur Institute of Guadeloupe and assigned specific shared international spoligotype signatures (SIT) according to the SITVIT2 data base [18 (link)].

To analyze the purity of the CTG repeat tract at the 3′ end CTG repeat array, TP-PCR was performed on both strands of the CTG repeat [32 (link),34 (link)]. The 3′ end CTG repeat was amplified using the primer downstream of the CTG repeat Somy4R-FAM (5′-FAM-CGG GTT TGG CAA AAG CAA ATT TCC CGA-3′), P3R (5′-TAC GCA TCC CAG TTT GAG ACG-3′) and P4CTG (5′-TAC GCA TCC CAG TTT GAG ACG TGC TGC TGC TGC TGC T-3′) primers as described in Tomé et al. [33 ]. Briefly, 20–100 ng of DNA from blood and trophoblast was amplified using 0.4 µM Somy4R-FAM and P3R primers and 0.04 µM P4CTG primer, 1× Custom master mix (Thermo Fisher Scientific, Courtaboeuf, France) and 0.06U Thermoperfect Taq polymerase (Peak International Products b.v, LZ Eerbeek, Netherlands)). The conditions of TP-PCR were as follows: denaturation at 94 °C for 5 min followed by 30 cycles at 94 °C for 1 min, 68 °C for 1 min 30 s and at 72 °C for 2 min and a final extension step at 72 °C for 10 min (1 cycle). The amplified product was analyzed using a 3500 XL genetic analyzer (Applied Biosystems, Foster City, CA, USA) and Gene Mapper software (Thermo Fisher Scientific, Courtaboeuf, France).