Table 1 Published pfhrp2/3 primer sequences, limits of detection, and optimized conditions
From: Streamlined, PCR-based testing for pfhrp2- and pfhrp3-negative Plasmodium falciparum
Assay # | Target | Primer sequences (5′→3′) | Reaction conditions | Cycling parameters | LOD,* ng/µL (genomes/µL) | Ref |
|---|---|---|---|---|---|---|
1 | pfhrp2 (exon 1/2) | Outer: For: GGTTTCCTTCTCAAAAAATAAAG Rev: TCTACATGTGCTTGAGTTTCG Optional—inner: For: GTATTATCCGCTGCCGTTTTTGCC Rev: CTACACAAGTTATTATTAAATGCGGAA | 200 nM each primer HotStarTaq MM (Qiagen, Venlo, Netherlands) 3 μL template DNA or 100 × diluted first-round product 25 μL reaction vol | 95 °C × 15 min; 40 cycles of 94 °C × 1 min, 50 °C × 1 min for outer primers or 55 °C × 1 min for inner primers, 60 °C × 1 min; 60 °C × 10 min | 10−5 (~ 0.4) | [3] |
2 | pfhrp2 (exons 1/2) | For: TATCCGCTGCCGTTTTTGCC Rev: AGCATGATGGGCATCATCCTA | 400 nM each primer HotStarTaq MM 3 μL template DNA 25 μL reaction vol | 95 °C × 15 min; 40 cycles of 94 ° × 1 min, 57 °C x 1 min, 60 °C × 1 min; 60 °C × 10 min | 10−1 (~ 4000) | [9] |
3 | pfhrp2 (exon 2) | For: ATTCCGCATTTAATAATAACTTGTGTAGC Rev: ATGGCGTAGGCAATGTGTGG | 400 nM each primer HotStarTaq MM 3 μL template DNA 25 μL reaction vol | 95 °C × 15 min; 40 cycles of 94 °C × 1 min, 59 °C × 1 min, 72 °C × 1 min; 72 °C × 10 min | 10−4 (~ 4) | [5] |
4 | pfhrp2 (exon 2) | Outer: For: CAAAAGGACTTAATTTAAATAAGAG Rev: AATAAATTTAATGGCGTAGGCA Optional—inner (hemi-nested): For: ATTATTACACGAAACTCAAGCAC Rev: AATAAATTTAATGGCGTAGGCA | 400 nM each primer HotStarTaq MM 3 μL template DNA or 100× diluted first-round product 25 μL reaction vol | 95 °C × 15 min; 40 cycles of 94 °C × 1 min, 57 °C × 1 min for outer primers or 62 °C for inner primers, 60 °C × 1 min; 60 °C × 10 min | 10−4 (~ 4) | [23] |
5 | pfhrp3 (exons 1/2) | For: TATCCGCTGCCGTTTTTGCTTCC Rev: TGCATGATGGGCATCACCTG | 400 nM primers HotStarTaq MM 3 μL template DNA 25 μL reaction vol | 95 °C × 15 min; 40 cycles of 94 °C × 1 min, 60 °C × 1 min, 60 °C × 1 min; 60 °C × 10 min | 10−4 (~ 4) | [9] |
6 | pfhrp3 (exon 2) | Outer: For: AATGCAAAAGGACTTAATTC Rev: TGGTGTAAGTGATGCGTAGT Optional—inner (hemi-nested): For: AAATAAGAGATTATTACACGAAAG Rev: TGGTGTAAGTGATGCGTAGT | 400 nM primers HotStarTaq MM 3 μL template DNA or 100 × diluted first-round product 25 μL reaction vol | 95 °C × 15 min; 40 cycles of 94 °C × 1 min, 55 °C × 1 min, 60 °C × 1 min; 60 °C × 10 min | 10−4 (~ 4) | [23] |
7 | pfldh (initial qPCR) | For: ACGATTTGGCTGGAGCAGAT Rev: TCTCTATTCCATTCTTTGTCACTCTTTC Probe: FAM-GTAATAGTAACAGCTGGATTTACCAAGGCCCCA-TAMRA | 200 nM primers 100 nM probe Probe Master qPCR Mix (Roche Diagnostics, Indianapolis, IN) 2 μL template DNA 12 μL reaction vol | 50 °C × 2 min; 95 °C × 10 min; 40 cycles of 95 °C × 15 s, 60 °C × 1 min | 10−4 (~ 4) | [31] |
8 | Pf -tubulin (confirmatory) | For: AATAAATCATAATGATGTGCGCAAGTGATCC Rev: AATAAATCATAATCCTTTGTGGACATTCTTCCTC | 300 nM primers FastStart Universal SYBR Green MM (Roche Diagnostics) 3 µL template DNA 25 µL reaction volume | 50 °C × 2 min; 95 °C × 10 min; 40 cycles of 95 °C × 15 s, 60 °C × 1 min; Dissociation analysis | 10−3 (~ 40) |