PCR-RFLP fbpC103/Ag85C103

The SNP fbpC¹⁰³,or Ag85C103, was described as a specific marker for the LAM lineage by Gibson et al. [4] in 2008. The PCR protocol used in this study was adapted as described by Vasconcelos et al. [7] in 2014. To perform the amplification, we used this set of primers: fbpC103 F and fbpC¹⁰³ R. The amplified products (519 bp) were analyzed on 2% agarose gel in 1× TBE. After this step, the enzymatic digestion was performed by restriction enzyme MnII (New England BioLabs Inc. USA). The MnII enzyme produces three restriction fragments in the amplified product: 365 bp, 96 bp, and 48 bp. The presence of SNP (G309A) (LAM) results in the loss of one of the three restriction sites [4, 7].