Table 2.
Sequences of applied primers and probes
| Test | Primer/probea | Sequence (5′- 3′)b | Nucleotide positionc | Amplicon sized |
|---|---|---|---|---|
| RLEP PCRe | RL - F2 | ACC TGA TGT TAT CCC TTG CAC | 39,741–39,761 | 167 bp |
| RL - R2 | CGC TAG AAG GTT GCC GTA TG | 39,908–39,889 | ||
| RLEP qPCR | RLEP - F | GCA GTA TCG TGT TAG TGA A | 39,839–39,857 | 69 bp |
| RLEP - R | CGC TAG AAG GTT GCC GTA TG | 39,908–39,889 | ||
| RLEP - P | 6FAM- CGC CGA CGG CCG GAT CAT CGA -BBQ | 39,885–39,865 | ||
| 16S rRNA RT qPCR | ML16S rRNA TaqF | GCA TGT CTT GTG GTG GAA AGC | 1,341,385–1,341,405 | 70 bp |
| ML16S rRNA TaqR | CAC CCC ACC AAC AAG CTG AT | 1,341,455–1,341,436 | ||
| ML 16S - TP2 | 6FAM- CCA TCC TGC ACC GCA AAA A -BBQ | 1,341,424–1,341,406 | ||
| GAPDHf (RT) qPCR | GAPDH fwd | GAA GGT GAA GGT CGG AGT C | 194–212 | 225 bp |
| GAPDH rev | GAA GAT GGT GAT GGG ATT TC | 419–400 | ||
| GAPDH TM | FAM-CAA GCT TCC CGT TCT CAG CCT -BBQ | 390–370 |
aF Forward primer, R Reverse primer, P/TP/TM Hydrolysis probes (TibMolBiol, Berlin, Germany)
b Hydrolysis probe with 6-Caboxyfluorescein fluorescent dye (6FAM) and BlackBerry Quencher (BBQ)
c Nucleotide positions are provided for the first copy of the respective amplicon in Mycobacterium leprae Br4923 (GenBank accession number FM211192.1). For GAPDH qPCR nucleotide positions are provided for the copy in Homo sapiens glyceraldehyde-3-phosphate dehydrogenase (GenBank accession number NM_002046.5) [25]
d bp = base pairs
e Direct DNA sequencing was conducted with the forward primer RL-F2. The sequence encompassed the region amplified by RLEP qPCR
f GAPDH = glyceraldehyde-3-phosphate-dehydrogenase