Table 1 Summary of PCR conditions used in the 18s-rRNA and COX-III PCR strategies for human malaria diagnosis

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