Table 3. Primers and probes.
| Primer/Probea | Sequence (5′–3′) | Target Geneb | Nucleotide Positionc | Amplicon Sized |
| MU16S TFMU16S TRMU16S TP | CGA TCT GCC CTG CAC TTC CCA CAC CGC AAA AGC TT6 FAM-CAC AGG ACA TGA ATC CCG TGG TC-BBQe | 16S rRNA | 4414800–44148174414718–44147344414740–4414762 | 100 bp |
| IS2404 TFIS2404 TRIS2404 TP2 | AAA GCA CCA CGC AGC ATC T AGC GAC CCC AGT GGA TTG6 FAM-CCG TCC AAC GCG ATC GGC A-BBQe | IS2404 | 96685–9666796627–9664496664–96646 | 59 bp |
| T13fT39f | TGC ACA CAG GCC ACA AGG GACG AAC GGG TGA GTA ACA CG | 16S rRNA | 4413906–44139254414822–4414840 | 935 bp |
Table 3 indicates primers and probes designed for the 16S rRNA RT-qPCR, the primers described by Fyfe et al., and a re-designed hydrolysis probe used for the amplification, detection, and quantification of IS2404 [9].
TF, forward primer; TR, reverse primer; TP2, hydrolysis probe (TibMolBiol, Berlin, Germany).
16S rRNA, gene for the ribosomal 16S RNA detected as 16S cDNA; IS2404, insertion sequence 2404.
Nucleotide positions are provided for the first (IS2404) or single (16S rRNA) copy of the respective amplicon in M. ulcerans Agy99 (GenBank accession no. CP000325.1) as determined by BLASTN analysis within GenBank (NCBI) [13].
bp, base pairs.
6 FAM, 6-Carboxyfluorescein fluorescent dye; BBQ, BlackBerry Quencher.
Primers T13 (forward) and T39 (reverse) were used for the amplification of a 935-bp region of the M. ulcerans 16S rRNA gene, encompassing the region amplified by qPCR primers MU16S TF and MU16S TR, to generate single copy replicates. Furthermore, these primers were used for sequencing of the M. ulcerans 16S rRNA gene (Table 1).