Table 1.
Analytical parameters of the RT-qPCR reactions.
| Reaction parameters | ||||
|---|---|---|---|---|
| Gene | Forward primer (5′-3′) (concentration in nM) | Reverse primer (5′-3′) (concentration in nM) | Probe (concentration in nM) | Amplicon size(bp) |
| Pfslarp | CCAAACACTCAGCACAGGAACA (500) | CCATACAGCCCTGGTATATAAATTA CTG (500) | FAM-ATGTCTATTGGCACTTACT- MGB (300) | 131 |
| Pfplp1 | CCTTTTAGGGTTTGGTATATCCTC TTC (200) | GAGCAGCTTTTCATTCCTGGT (200) | HEX-TCAGGGAGAATCAATTC- MGB (250) | 96 |
| Pfcsp | TCAACTGAATGGTCCCCATGT (100) | GAGCCAGGCTTTATTCTAACTTGAA T (200) | Cy5-TGTAACTTGTGGAAATGG- MGB-(250) | 68 |
| Quality control | ||||
| Gene | Reaction Efficiency (%) | Linearity (R 2 ) | Dynamic Range (Ct) | |
| Pfslarp | 108.5 | 0.9992 | 24.0–34.0 | |
| Pfplp1 | 99.70 | 0.9969 | 26.0–36.0 | |
| Pfcsp | 95.5 | 0.9996 | 16.0–30.0 | |
| % CV of RQ units | Multiplexing | |||
| Pfslarp | 19.9 | 2-plex (A): Pfplp1 + Pfcsp | 2-plex (B): Pfslarp + Pfcsp | |
| Pfplp1 | 9.8 | No change in sensitivity | Loss of sensitivity for Pfslarp (+1.5 Ct shift) | |