Table A2.
Reaction mixes and run conditions for the beta-lactamase real-time PCR assays, part 1 out of 2.
| blaCTX-M Gene-Specific Assay | blaVIM, blaIMP, blaNDM and blaKPC Gene-Specific Assay | blaOXA-23-like, blaOXA-40/24-like, blaOXA-48-like and blaOXA-58-like Gene-Specific Assay | |
|---|---|---|---|
| Reaction chemistry | |||
| Master Mix | HotStarTaq (Qiagen) | HotStarTaq (Qiagen) | HotStarTaq (Qiagen) |
| Reaction volume (µL) | 20.0 | 20.0 | 20.0 |
| Forward primer concentration (nM) | 320.0 (each) | 750.0 (each) | 800.0 (OXA-48-like & OXA-40/24-like), 600.0 (OXA-58-like), 400.0 (OXA-48-like) |
| Reverse primer concentration (nM) | 320.0 (each) | 375 (IMP reverse primers I & II), 750 (all others) | 800.0 (OXA-48-like & OXA-40/24-like), 600.0 (OXA-58-like), 400.0 (OXA-48-like) |
| Probe concentration (nM) | 160.0 | 188.0 (VIM), 200.0 (both IMP probes), 250 (NDM & KPC) | 400.0 (OXA-48-like & OXA-40/24-like), 250.0 (OXA-58-like), 150.0 (OXA-48-like) |
| Final Mg2+ concentration (nM) | 3.0 | 3.0 | 6.0 |
| Bovine serum albumin (ng/µL) | 2.0 | 2.0 | 2.0 |
| Run conditions | |||
| Initial denaturation | 95 °C, 15 min | 95 °C, 15 min | 95 °C, 15 min |
| Cycle numbers | 40 | 40 | 40 |
| Denaturation | 95 °C, 15 s | 95 °C, 15 s | 95 °C, 15 s |
| Annealing | Combined with amplification | Combined with amplification | Combined with amplification |
| Amplification | 60 °C, 60 s | 60 °C, 60 s | 60 °C, 60 s |
| Hold | 40 °C, 20 s | 40 °C, 20 s | 40 °C, 20 s |