Volume 12, Number 3—March 2006
Research
Identifying and Quantifying Genotypes in Polyclonal Infections due to Single Species
Table 1
Primers and probes used for real-time polymerase chain reaction*
Primer/probe sequence | Sizes (bp) | Tm (°C) | Fluorophore, quencher | Final reactant concentration (nmol/L) | |
---|---|---|---|---|---|
Parasites with K1 and hybrid sequences in block 2 of msp1 | |||||
K1F 5´-AGGTGCAAGTGCTCAAAGTG-3´ | 108–171 | 50.3 | Texas Red, BHQ-2 | 100 | |
K1R 5´-CACCAGATGAAGTATTTGAACG-3´ | 49.2 | 100 | |||
PROBE: 5´-AAGTGGTACAAGTCCATCATCTCGT-3´ | 54.9 | 300 | |||
Hybrid F 5´-GAAGGAACAAGTGGAACAGC-3´ | 96–168 | 48.7 | Cy5, BHQ-2 | 200 | |
Hybrid R 5´-GCAGCACCTGGAGATCTTATA-3´ | 48.8 | 200 | |||
PROBE: 5´-TTCACTTATTTCCCCATGAGCCCC-3´ | 54.2 | 400 | |||
Primers for positive control (EBA175) | |||||
EBA175 F 5´-GGTTATTCAACTAAGGCAGAA-3´ | 95 | 46.0 | Cy 5, BHQ-3 | 100 | |
EBA175 R 5´-TCCACCATTCTTTTCTAAAATTTT-3´ | 50.6 | 100 | |||
PROBE: 5´-TCATTTCCCATAGCAAGATGTCC | 60.0 | 100 | |||
LUX primers (MAD 20, RO33) | |||||
MAD20 F 5´-AATGAAGGAACAAGTGGAAC-3´ | 52–205 | 48.7 | FAM | 200 | |
MAD20 R 5´-GAATTATCTGAAGGATTTGTACG-3´ | 47.4 | (reverse) | 200 | ||
RO33 F 5´-GCAGATGCTGTAAGTACTCAA-3´ | 148 | 44.8 | JOE | 200 | |
RO33 R 5´-GCAGCACCTGGAGATCTTATA-3´ | 44.8 | (forward) | 200 |
*msp1, merozoite surface protein 1, EBA175, erythrocyte-binding antigen 175; Tm, melting temperature.