Approach | Primers | Reagents final concentration | Cycling parameters |
---|---|---|---|
18s rRNA genus specific DIRECT PCR nest 1 | rPLU1 TCAAAGATTAAGCCATGCAAGTGA rPLU5 CCTGTTGTTGCCTTAAACTTC | 1X blood phusion buffer, 1 mM of primers, 1.0 mM of MgCl2 and 0.25 µL of phusion blood II DNA polymerase | 98 °C for 4 min; 30 cycles of 94 °C for 1 min, 65° for 2 min and 72 °C for 2 min, and 72 °C for 4 min |
18s rRNA genus specific PCR nest 2 | rPLU3 TTTTTATAAGGATAACTACGGAAAAGCTGT rPLU4 TACCCGTCATAGCCATGTTAGGCCAATACC | 1X PCR buffer, dNTPmix (200 uM each), 1 mM of primers, 1.0 mM of MgCl2 50 mM and 0.15 µL of Taq DNA polymerase | 94 °C for 4 min; 35 cycles of 94 °C for 30 s, 62° for 1 min and 72 °C for 1 min, and 72 °C for 4 min |
18s rRNA species specific P. vivax nest 2 | rVIV1 CGCTTCTAGCTTAATCCACATAACTGATAC rVIV2 ACTTCCAAGCCGAAGCAAAGAAAGTCCTTA | 1X PCR buffer, dNTPmix (200 uM each), 1 mM of primers, 1.0 mM of MgCl2 50 mM and 0.15 µL of Taq DNA polymerase | 94 °C for 4 min; 35 cycles of 94 °C for 30 s, 58° for 1 min and 72 °C for 1 min, and 72 °C for 4 min |
18s rRNA species specific P. falciparum nest 2 | rFAL1 TTAAACTGGTTTGGGAAAACCAAATATATT rFAL2 ACACAATGAACTCAATCATGACTACCCGTC | 1X PCR buffer, dNTPmix (200 uM each), 1 mM of primers, 1.0 mM of MgCl2 50 mM and 0.15 µL of Taq DNA polymerase | 94 °C for 4 min; 35 cycles of 94 °C for 30 s, 58° for 1 min and 72 °C for 1 min, and 72 °C for 4 min |
COX-III DIRECT PCR nest1 | Plas_COX3_F TGATAGCGGTTAACCTTTCYTTTTCCTTACG Plas_COX3_R CTGTTATCCCCGGCGAACCTTCTTAC | 1X blood phusion buffer, 1 mM of primers, and 0.25 µL of phusion blood II DNA polymerase | 98 °C for 4 min; 35 cycles of 98 °C for 1 s, 70° for ×5 s and 72 °C for 30 s, and 72 °C for 2 min |
COX-III nest2 | shortCOX-III_F AGCGGTTAACCTTTC T TTTTCCTTACG shortCOXIII_R AGTGCATCATGTATGACAGCATGTTTACA | 1X PCR buffer, dNTPmix (200 uM each), 0.5 mM of primers, 1.5 uM of MgCl2 50 mM and 0.2 µL of Taq DNA polymerase | 94 °C for 5 min; 40 cycles of 94 °C for 1 min, 62° for 1 min and 72 °C for 90 s, and 72 °C for 10 min |
COX-III Single DIRECT PCR | shortCOXIII_F AGCGGTTAACCTTTC T TTTTCCTTACG shortCOXIII_R AGTGCATCATGTATGACAGCATGTTTACA | 1X blood phusion buffer, 1 mM of primers, and 0.30 µL of phusion blood II DNA polymerase | 98 °C for 4 min; 70 cycles of 98 °C for 1 s, 70° for ×5 s and 72 °C for 35 s, and 72 °C for 10 min |