Background
Methods
Clinical samples
Plasmodium falciparum laboratory strain samples
SNP selection and assay design
SNP Assay Name | Primers/Probes | Sequence (5'-3') |
---|---|---|
DHFR16 | Forward | CAAGTCTGCGACGTTTTCGATATTT |
Reverse | CCTCATTTTTTTTCCCCTCATTTTTGC | |
Probe1 | CCTTACAACATGCACATAT | |
Probe2 | AACCTTACAACATACACATAT | |
DHFR22 | Forward | GCGACGTTTTCGATATTTATGCCATA |
Reverse | TCCTAGACCTCTAAATGTGTAGTTATTAAAAACCT | |
Probe1 | CCCTCATTTTTGCTTTCAA | |
Probe2 | CCCTCATTTTTGTTTTCAA | |
DHFR51 | Forward | ACTACACATTTAGAGGTCTAGGAAATAAAGGA |
Reverse | GTTGTAACTGCACAAAAATATTTCATATCTAGGG | |
Probe1 | CCATGGAAATGTAATACCAT | |
Probe2 | CCATGGAAATGTATTACCAT | |
DHFR59 | Forward | CTAGGAAATAAAGGAGTATTACCATGGAAATGT |
Reverse | CATCTCTTATATTTCAATTTTTCATATTTTGATTCATTCA | |
Probe1 | CCCTCATTTTTGCTTTCAA | |
Probe2 | CCCTCATTTTTGTTTTCAA | |
DHFR108 | Forward | ATGTAAATGATATGCCTAATTCTAAAAAATTACAAAATGT |
Reverse | GACAATATAACATTTATCCTATTGCTTAAAGGT | |
Probe1 | CTTTCCCAGCTTGTTCT | |
Probe2 | CTTTCCCAGTTTGTTCT | |
DHFR164 | Forward | ACAAAGTTGAAGATCTAATAGTTTTACTTGGGAAA |
Reverse | TTAATTTCTTTTCTAAAAATTCTTGATAAACAACG | |
Probe1 | CCCTCATTTTTGCTTTCAA | |
Probe2 | CCCTCATTTTTGTTTTCAA | |
MDR86 | Forward | AGGAGGAACATTACCTTTTTTTATATCTGTGT |
Reverse | ATTGTACTAAACCTATAGATACTAATGATAATATTATAGGAT | |
Probe1 | CATCACCTAAATTCATGTTC | |
Probe2 | CATCACCTAAATACATGTTC | |
MDR184 | Forward | GGTACGAAATTTATAACAATTTTTACATATGCCAGTT |
Reverse | AAAAACGCAAGTAATACATAAAGTCAAACGT | |
Probe1 | CCTTTTTAGGTTTATATATTTG | |
Probe2 | CCTTTTTAGGTTTATTTATTTG | |
MDR1034 | Forward | GACAAAAAAGAAGAATTATTGTAAATGCAGCTT |
Reverse | AGGATCCAAACCAATAGGCAAAACT | |
Probe1 | CTTTGACTGAATCCC | |
Probe2 | TTTGACAGAATCCC | |
MDR1042 | Forward | GGATTCAGTCAAAGCGCTCAATTA |
Reverse | GTACCTCTTTTAATTAAGAAGGATCCAAACCA | |
Probe1 | ATAGGCAAAACTATTAATAAA | |
Probe2 | TAGGCAAAACTATCAATAAA | |
DHPS581 | Forward | TTCTTGTATTAAATGGAATACCTCGTTATAGGAT |
Reverse | TATACATGTATATTTTGTAAGAGTTTAATAGATTGATCATG | |
Probe1 | TTTCTTCGCAAATCC | |
Probe2 | TTTCTTCCCAAATCC |
SNP assay
DNA amplification and cloning PCR fragments into TOPO TA vector
Plasmid generated | Sequence (5'-3') | PCR fragment size | |
---|---|---|---|
16-3D7/7G8 | Forward Primer | CAAGTCTGCGACGTTTTCGATATTT | |
Reverse Primer | TTAATTTCTTTTCTAAAAATTCTTGATAAACAACG | 526 bp | |
86-K1/7G8 | Forward Primer | TGGGTAAAGAGCAGAAAGAGAAA | |
Reverse Primer | TTGCAACAGTTCTTATTCCCATT | 735 bp | |
1034-K1/7G8 | Forward Primer | CAAGCGGAGTTTTTGCATTT | |
Reverse Primer | TTCCACCATCATCTCTTACATCA | 447 bp | |
581-K1/7G8 | Forward Primer | TGCATAAAAGAGGAAATCCACA | |
Reverse Primer | TCCAATTGTGTGATTTGTCCA | 357 bp |
Sequencing
Results
SNP assays performance
Codon | 16 | 22 | 51 | 59 | 108 | 164 | 86 | 184 | 1034 | 1042 | 581 |
---|---|---|---|---|---|---|---|---|---|---|---|
Wild-type | C | G | A | T | G | A | A | A | A | A | C |
Mutant |
T
|
A
|
T
|
C
|
A
|
T
|
T
|
T
|
T
|
G
|
G
|
3D7 | C | G | A | T | G | A | A | A | A | A | C |
7G8 | C | G |
T
| T |
A
| A | A |
T
|
T
|
G
| C |
K1 | C | G | N | N |
A
| A |
T
| A | A | A |
G
|