Table 1 Primers sequences and real-time PCR conditions used to detect Plasmodium species
From: An innovative tool for moving malaria PCR detection of parasite reservoir into the field
Real-time PCR name | Primer name | Sequence (5′-3′) | Master mix | Assay parameters | Melt parameters | T° melt peak | |
|---|---|---|---|---|---|---|---|
Real-time PCR screening | RTPCRScreening2_F | TGGAGTGGATGGTGTTTTAGA | Hot FirePol EvaGreen qPCR Mix Solis Biodyne 1X (#08-24-00020), Primers 150 nM, 5 μl DNA template, Total volume 20 μl | 95°C-15 min 45 cycles: 95°C-15 sec/60°C-20 sec/72°C-20 sec 95°C-2 min 68°C-2 min | From 68 to 90°C, increment 0.2°C for 0.05 sec | 76.4-78.4°C | |
RTPCRScreening2_R | TTGCACCCCAATARCTCATTT | ||||||
Nested real-time PCR species | Primary PCR | RTPCRScreening2_F | TGGAGTGGATGGTGTTTTAGA | Hot FirePol DNA Pol. Solis BioDyne 1.25 U (#01-02-01000), dNTP 200 μM, MgCl2 2.5 mM, Primers 250 nM, 5 μl DNA template. Total volume 20 μl. | 94°C -15 min 20 cycles: 94°C-30 sec/ 58°C -1 min/ 72°C-1 min 72°C-10 min | N/A | N/A (PCR product size: 400 bp) |
RTPCRSreening3_R | ACCCTAAAGGATTTGTGCTACC | ||||||
Nested real-time PCR Pf | Pf_RTPCR_F | ATGGATATCTGGATTGATTTTATTTATGA | Hot FirePol EvaGreen HRM Mix Solis Biodyne 1X (#08-33-00001), Primers 250 nM, 5 μl template Primary PCR products 1:10, Total volume 20 μl | 95°C-15 min 35–45 cycles*: 95°C-10 sec/62°C-20 sec/72°C-25 sec 95°C-1 min 40°C-1 min | From 65 to 90°C, increment 0.2°C for 0.05 sec. | 78.6-79.6°C | |
Pf_RTPCR_R | TCCTCCACATATCCAAATTACTGC | ||||||
Nested Real-time PCR Pv | Pv_RTPCR_F | TGCTACAGGTGCATCTCTTGTATTC | 74.8-75.8°C | ||||
Pv_RTPCR_R | ATTTGTCCCCAAGGTAAAACG | ||||||
Nested real-time PCR Pm | Pm_RTPCR_F | ACAGGTGCATCACTTGTATTTTTTC | 75.4-76.4°C | ||||
Pm_RTPCR_R | TGCTGGAATTGAAGATAATAAATTAGTAATAACT | ||||||
Nested Real-time PCR Po | Po_RTPCR_F | GTTATATGGTTATGTGGAGGATATACTGTT | 73.2-74.2°C | ||||
Po_RTPCR_R | CGAATGGAAGAATAAAATGTAGTACG | ||||||