Table 3.
Real-time PCR-HRM assay for M. leprae DRDR mutation detection (DRDR assays)
| Sample typea | Template | Assay resultb |
||||||||
|---|---|---|---|---|---|---|---|---|---|---|
|
folP1 |
rpoB |
gyrA |
||||||||
| Reported DRDR genotype | HRM cluster | HRM cluster verification by sequencing | Reported DRDR genotype | HRM cluster | HRM cluster verification by sequencing | Reported DRDR genotype | HRM cluster | HRM cluster verification by sequencing | ||
| ADML | NHDP63 | No mutation | WT | No mutation | WT | No mutation | WT | No mutation | ||
| MFP | Airaku-2c | P(CCC)55L(CTC) | WT | No mutation | S(TCG)456L(TTG) | WT | No mutation | No mutation | WT | No mutation |
| Airaku-3 | T(ACC)53I(ATC) | V | No mutation | WT | No mutation | WT | ||||
| Amami | P(CCC)55L(CTC) | V | No mutation | WT | No mutation | WT | ||||
| Hoshizuka-4 | P(CCC)55S(CTC) | V | S(TCG)456L(TTG) | V | A(GCA)91V(GTA) | V | ||||
| Kusatsu-3 | T(ACC)53I(ATC) | V | No mutation | WT | No mutation | WT | ||||
| Kusatsu-6 | P(CCC)55L(CTC) | V | D(GAT)441Y(TAT) | V | No mutation | WT | ||||
| Ryukyu-6 | No mutation | WT | No mutation | WT | A(GCA)91V(GTA) | V | ||||
| Zensho-2 | P(CCC)55L(CTC) | V | No mutation | WT | No mutation | WT | ||||
| Zensho-4d | T(ACC)53I(ATC) | V | S(TCG)456L(TTG) | V | A(GCA)91V(GTA) | V | A(GCA)91V(GTA) and no mutation | |||
| Zensho-5e | P(CCC)55L(CTC) | V | P(CCC)55L(CTC), T(ACC)53I(ATC), and no mutation | S(TCG)456L(TTG) | V | S(TCG)456L(TTG) | No mutation | V | No mutation; A(GCA)91V(GTA) | |
| Zensho-9 | No mutation | V | P(CCC)55L(CTC) | H(CAC)451Y(TAC) | V | H(CAC)451Y(TAC) | No mutation | WT | ||
| Zensho-15 | Unknown | V | P(CCC)55L(CTC) | Unknown | V | S(TCG)456L(TTG) | Unknown | V | A(GCA)91V(GTA) | |
| Gushiken | No mutation | WT | No mutation | WT | No mutation | WT | ||||
| Hoshizuka-5 | No mutation | WT | No mutation | WT | No mutation | WT | ||||
| Indonesia-1 | No mutation | WT | No mutation | No mutation | WT | WT | No mutation | WT | No mutation | |
| Korea-3-2 | No mutation | WT | No mutation | WT | No mutation | WT | ||||
| Thai-53 | No mutation | WT | No mutation | WT | No mutation | WT | ||||
| Thai-311 | No mutation | WT | No mutation | WT | No mutation | WT | ||||
ADML, armadillo-derived M. leprae; MFP, mouse footpad-derived M. leprae.
Reported DRDR genotype sequence data were verified by the method of Matsuoka (22). HRM cluster data are designated WT for wild-type target sequences or V for variant target sequences. NHDP63 strains with the same sequences as those of the TN strain were considered WT. For HRM cluster verification by sequencing, representative samples of each of the clusters were verified by PCR product sequencing, and the genotypes detected are indicated.
The HRM clustering results were not concordant with expected genotypes for both the rpoB and folP1 genes for strain Airaku-2 (22). VNTR strain typing performed for this strain confirmed that it was indeed not Airaku 2 (44). However, the designation Airaku-2 was retained during the course of the study and in all tables in this report.
The HRM assay for gyrA DRDR separated this strain from the wild type and the other mutants; DNA sequencing results showed a mixed allele (C and T) (see Fig. 2 [gyrA; orange curves] and Fig. 3).
The HRM assay for folP1 and gyrA DRDRs separated this strain from the wild type and the other mutants that share the same genotype; DNA sequencing showed minor mixed alleles at codons 53 and 55 in folP1 and codon 91 in gyrA (see Fig. 2 [blue curve in folP1 panel B and orange curve in gyrA panel B] and Fig. 3).