Table A4.
Reaction mixes and run conditions for the sexually transmitted infections PCRs and Schistosoma spp.-specific PCR assays.
| Chlamydia trachomatis Screening and Differentiation Assay | Neisseria gonorrhoeae, Mycoplasma genitalium and Treponema pallidum Assay | Schistosoma haematobium Complex and Schistosoma mansoni Complex Assay | |
|---|---|---|---|
| Reaction chemistry | |||
| Master Mix | HotStarTaq (Qiagen) | HotStarTaq (Qiagen) | HotStarTaq (Qiagen) |
| Reaction volume (µL) | 20.0 | 20.0 | 20.0 |
| Forward primer concentration (nM) | 50.0 (screening), 600.0 (differentiation) | 900.0 (T. pallidum), 1000.0 (M. genitalium), 400.0 (gonococci, both assays) | 500.0 (each) |
| Reverse primer concentration (nM) | 100.0 (screening), 600.0 (differentiation) | 900.0 (T. pallidum), 1000.0 (M. genitalium), 400.0 (gonococci, both assays) | 500.0 (each) |
| Probe concentration (nM) | 100.0 (screening), 200.0 (differentiation) | 250.0 (T. pallidum), 225.0 (M. genitalium), 160.0 (gonococci, both assays) | 300.0 (each) |
| Final Mg2+ concentration (nM) | 4.0 | 5.0 | 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 | 45 | 50 | 40 |
| Denaturation | 95 °C, 15 sec. | 95 °C, 15 sec. | 95 °C, 15 sec. |
| Annealing | Combined with amplification | Combined with amplification | Combined with amplification |
| Amplification | 60 °C, 60 sec. | 60 °C, 60 sec. | 65 °C, 60 sec. |
| Hold | 40 °C, 20 sec. | 40 °C, 20 sec. | 40 °C, 10 sec. |
Min. = minute, sec. = second.