Table A3.
Reaction mixes and run conditions for the beta-lactamase real-time PCR assays, part 2 out of 2.
| blaGES, blaNMC_A/IMI, blaBIC and blaSME Gene-Specific Assay | blaGIM Gene-Specific Assay | blaDIM 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) | 750.0 (each) | 400 | 400 |
| Reverse primer concentration (nM) | 750.0 (each) | 400 | 400 |
| Probe concentration (nM) | 375 (NMC_A/IMI), 188 (all others) | 200 | 250 |
| Final Mg2+ concentration (nM) | 3.0 | 6.0 | 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, 30 s | 95 °C, 30 s |
| Annealing | Combined with amplification | 46 °C, 40 s | 46 °C, 40 s |
| Amplification | 60 °C, 60 s | 72 °C, 50 s | 72 °C, 50 s |
| Hold | 40 °C, 20 s | 40 °C, 20 s | 40 °C, 20 s |