| Reagents | Quantity | Final concentration |
| 1. Thermopol buffer (10×) | 2.5 µL | 1× |
| 2. MgSO4 (100 mM) | 2 µL | 8 mM |
| 3.dNTP mix (20 mM) | 1.75 µL | 1.4 mM |
| 4. F3 primer | 0.5 µL | 0.2 mM |
| 5. B3 primer | 0.5 µL | 0.2 mM |
| 6. FIP primer (F1c and F2) | 1.25 µL | 1.6 µM |
| 7. BIP primer (B1c and B2) | 1.25 µL | 1.6 µM |
| 8. FLP primer | 0.625 µL | 0.8 µM |
| 9. BLP primer | 0.625 µL | 0.8 µM |
| 10. Betaine (5 M) | 2 µL | 0.4 mM |
| 11. Bst polymerase (8000 U/ml) | 1 µL | 320 U/mL |
| 12. Reverse transcriptase (200 U/ml) | 0.1 µL | 8 U/mL |
| 13. EVA Green (20×) | 1 µL | 1× |
| 14. HNB dye | 1.5 µL | 120 µM |
| 15. Sample | 5 - 10 µL |
|
| 16. PCR grade water | Up to 25 µL |
|