Table 1.
PCR conditions for the 18s-rRNA nested-PCR and the new COX-I PCRs for Plasmodium sporozoite detection
| Diagnostic description | Reagents quantities and final concentration | Thermal profile |
|---|---|---|
| 18s-rRNA genus specific PCR nest-1 [19] |
1X PCR buffer, 80 μM dNTPmix, 0.8 mM MgCl2, 0.1 mM each primer (rPLU1–rPLU5), 0.25 U Taq polymerase | 94 °C for 4 min; 35 cycles of 94 °C for 30 s, 55 °C for 1 min, 72 °C for 1 min; and 72 °C for 4 min. Time: 120 min |
| 18s-rRNA genus specific PCR nest-2 [19] |
Same as nest-1 but using rPLU3–rPLU4 primers | Same as nest-1 but annealing temperature is 62 °C. Time: 120 min |
| Conventional COX-I PCR | 1X PCR buffera, 10 mM dNTPs, 0.4 mM each primer, 1.5 mM MgCl2 and 0.2 µL of recombinant Taq polymerase | 94 °C for 5 min; 40 cycles of 94 °C for 1 min, 62 °C for 1 min, 72 °C for 90 s; and 72 °C for 10 min. Time: 155 min |
| Fast COX-I PCR | 1X blood phusion buffera, 1 mM each primer, and 0.125 µL of blood phusion polymerase | 98 °C for 4 min; 70 cycles of 98 °C for 1 s, 69 °C for 5 s, 72 °C for 35 s; and 72 °C for 10 min. Time: 62 min |
aThe Blood Phusion buffer contains MgCl2 at a final concentration of 3 mM