TABLE 1.
Primer sequences and nucleotide positions of rpoB PCR and sequencing primersa
| Assay | Primerb | Sequence (5′ to 3′)c | Nucleotide positiond | Amplicon size, bpe |
|---|---|---|---|---|
| Af | MF | CGA CCA CTT CGG CAA CCG | 785041–785058 | 351 |
| MR | TCG ATC GGG CAC ATC CGG | 785374–785391 | ||
| Bg | MuB-F | CAT CAC CAG CTC GAC GCT | 784912–784929 | 606 |
| MuB-R | TGG ATC TCG TCG GAA ACG | 785500–785517 | ||
| Bseek-F | AAT ACC TGG TCC GCT TGC | 784959–784976 |
Shown are the primer sequences, nucleotide positions within the M. ulcerans genome, and corresponding amplicon sizes. The rpoB gene encodes the beta subunit of (myco)bacterial RNA polymerases. Significant sequence concordances of primers with human or other (myco)bacterial DNA were excluded by Primer BLAST (PubMed, NCBI).
F, forward primer; R, reverse primer. Primers MF and MR were used in assay A for the amplification of a 351-bp fragment of the M. ulcerans rpoB gene (including the RRDR) encompassing the region sequenced by primer MF. Primers MuB-F and MuB-R were used in assay B for the amplification of a 606-bp fragment of the M. ulcerans rpoB gene (including the RRDR) encompassing the region sequenced by primer Bseek-F.
Primer sequence from the 5′ to the 3′ end.
Nucleotide positions are provided for the respective amplicon in M. ulcerans strain Agy99 (GenBank accession no. CP000325 [PubMed, NCBI]).
Amplicon sizes for rpoB PCR of assay A or B, respectively.
For assay A, final concentrations of PCR reagents per 20-μl reaction: 0.5 μM each primer (TIB-Molbiol, Berlin, Germany); 2.5 mM MgCl2, 0.8 mM deoxynucleoside triphosphates (dNTPs), 0.05 U/μl AmpliTaq Gold DNA polymerase, 1× PCR buffer II (Applied Biosystems, Foster City, CA); template DNA, 2 μl; run protocol, 95°C for 5 min, 37 cycles at 95°C for 15 s, 56°C for 15 s, and 72°C for 30 s, and final extension at 72°C for 5 min.
For assay B, final concentrations of PCR reagents per 20-μl reaction: 0.3 μM each primer (TIB-Molbiol); 1.5 mM MgSO4, 0.8 mM dNTPs, 0.02 U/μl KOD Hot Start polymerase, 1× PCR buffer for KOD (Merck, Darmstadt, Germany); template DNA, 2 μl; run protocol, 95°C for 2 min and 39 cycles at 95°C for 20 s, 63°C for 10 s, and 70°C for 15 s.