Two-tube real-time PCR assay. Assay I, left plots (A1 to F1), determines if the sample contains the wild-type sequences for the 23S rrl gene at position 2058-2059 (green curves), if the sample contains M. abscessus subsp. bolletii (red curves), and if the sample contains the erm(41) T28C mutation (blue curves). Assay II, right plots (A2 to F2), determines if the sample contains M. abscessus subsp. abscessus (green curves) or M. abscessus subsp. massiliense (blue curves) and if the sample contains the erm(41) deletion (red curves). The following six different samples are shown: (A) M. abscessus subsp. abscessus, with a wild-type 23S rrl gene at position 2058-2059, no erm(41) T28C mutation, and no erm(41) deletion; (B) M. abscessus subsp. abscessus, with a wild-type 23S rrl gene at position 2058-2059, but with an erm(41) T28C mutation and no erm(41) deletion; (C) M. abscessus subsp. abscessus, with a mutation in the 23S rrl gene at position 2058-2059, no erm(41) T28C mutation, and no erm(41) deletion; (D) M. abscessus subsp. bolletii with a wild-type 23S rrl gene at position 2058-2059, no erm(41) T28C mutation, and no erm(41) deletion; (E) M. abscessus subsp. massiliense with a wild-type 23S rrl gene at position 2058-2059, no erm(41) T28C mutation, and no erm(41) deletion; (F) M. abscessus subsp. massiliense with a wild-type 23S rrl gene at position 2058-2059, no erm(41) T28C mutation, but with a deletion in the erm(41) gene.