Background
Methods
Sample collection
Extraction of DNA
High resolution melting analysis
Primer | Target | Sequence | Fragment size (bp) | HRM temperature range (°C) |
---|---|---|---|---|
IS6110-F | TCCGGCCGGTGGTCG | |||
IS6110
| 103 | |||
IS6110-R | CGGCGCTTGTGGGTCAA | |||
rpoB-F | CGCGATCAAGGAGTTCTTC | |||
rpoB
| 118 | |||
rpoB-R | TGACAGACCGCCGGGCCC | 80–95 | ||
katG-F | GCTGAGCCAATTCATGGACC | |||
katG
| 235 | |||
katG-R | TGACAGACCGCCGGGCCC | |||
inhA-F | GCGGTCACACTTTCGGTAA | |||
Promoter inhA
| 180 | |||
inhA-R | GGTGTTCGTCCATACGACCT |
DNA sequencing
Primer | Gene | Sequence | Fragment size (bp) |
---|---|---|---|
rpoB-FS | TGGTCCGCTTGCACGAGGGTCAGA | ||
rpoB
| 758 | ||
rpoB-RS | CAGGAAGGGAATCATCGCGG | ||
katG-FS | TGCAGATGGGGCTGATCTACG | ||
katG
| ACCCATGTCTCGGTGGATCAG | 596 | |
katG-RS | |||
inhA-FS | ACATACCTGCTGCGCAAT | ||
inhA
| TCACATTCGACGCCAAAC | 400 | |
inhA-RS |
Statistical analysis
Results and Discussion
Detection of rifampicin resistance using HRM analysis
Detection of isoniazid resistance using HRM analysis
DNA sequencing
DRUG | PHENOTYPE | TARGET | N° | MUTATION | HRM CURVE ASSAY | DNA SEQUENCING |
---|---|---|---|---|---|---|
RIFAMPICIN | Resistant (78) |
rpoB
| 53 | S531L (TCG → TTG) | mutant | mutant |
14 | D516V (GAC → GTC) | mutant | mutant | |||
2 | H526D (CAC → GAC) | mutant | mutant | |||
1 | Q513L (CAA → CTA) | mutant | mutant | |||
1 | Q513P (CAA → CCA) | mutant | mutant | |||
1 | M515I (ATG → ATA), D516G (GAC → GGC) | mutant | mutant | |||
1 | D516Y (GAC → TAC), N518H (AAC → CAC) | mutant | mutant | |||
1 | H526R (CAC → CGC) | mutant | mutant | |||
1 | H526Y (CAC → TAC) | mutant | mutant | |||
1 | S531F (TCG → TTT) | mutant | mutant | |||
1 | S531W (TCG → TGG) | mutant | mutant | |||
1 | No mutation | wild | wild | |||
Susceptible (89) | 87 | Not sequenced | wild | - | ||
2 | 514 Phe (TTC → TTT) | mutant | wild | |||
ISONIAZID | Resistant (78) |
katG
| 66 | S315T (AGC → ACC) | mutant | mutant |
1 | S315T (AGC → ACC), C → T (−15) | mutant | mutant | |||
inhA
| 10 | C → T (−15) | mutant | mutant | ||
katG,inhA
| 1 | No mutation | wild | wild | ||
Susceptible (89) | 87 | Not sequenced | wild | - | ||
2 | No mutation | wild | wild |
Analysis of sensitivity and specificity of HRM analysis
Drug | Target | HRM profile | % variant | Drug susceptibility | % resistant | Sensitivity (%) | Specificity (%) | ||
---|---|---|---|---|---|---|---|---|---|
+ | − | + | − | ||||||
Rifampicin |
rpoB
| 79 | 88 | 47.3 | 78 | 89 | 46.7 | 98.7 (92.2–99.9) | 97.75 (91.2–99.6) |
Isoniazid |
katG and/or | 77 | 90 | 46.1 | 78 | 89 | 46.7 | 98.7 (92.2–99.9) | 100 (94.2–99.9) |
inhA promoter |