Real-time PCR name | Primer name | Sequence (5'-3') | Master mix | Assay parameters | Melt Parameters | T° Melt peak | |
---|---|---|---|---|---|---|---|
"Screening" | RTPCRScreening2_F | TGGAGTGGATGGTGTTTTAGA | Hot FirePol EvaGreen qPCR Mix Solis Biodyne 1X (#08- 24-00020), Primers 250 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. | 76.2 - 78.8°C | |
RTPCRScreening2_R | TTGCACCCCAATARCTCATTT | ||||||
Plasmodium sp. identification | 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 min3. 72°C-10 min. | N/A | N/A |
RTPCRSreening3_R | ACCCTAAAGGATTTGTGCTACC | ||||||
Pf real-time PCR | Pf_RTPCR_F | ATGGATATCTGGATTGATTTTA TTTATGA | Hot FirePol EvaGreen HRM Mix Solis Biodyne 1X (#08- 33-00001), Primers 250 nM, 5 μl template Primary PCR products 1:1000, total volume 20 μl | 95°C-15 min. 40 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. | 78.8-79.6°C | |
Pf_RTCPR_R | TCCTCCACATATCCAAATTAC TGC | ||||||
Pv real-time PCR | Pv_RTPCR_F | TGCTACAGGTGCATCTCTTG TATTC | 75.2-76°C | ||||
Pv_RTPCR_R | ATTTGTCCCCAAGGTAAAACG | ||||||
Pm real- time PCR | Pm_RTPCR_F | ACAGGTGCATCACTTGTATTTTTTC | 75.8-76.2°C | ||||
Pm_RTPCR_R | TGCTGGAATTGAAGATAATAAATT AGTAATAACT | ||||||
Po real- time PCR | Po_RTPCR_F | GTTATATGGTTATGTGGAGGATA TACTGTT | 73.4-74.2°C | ||||
Po_RTPCR_R | CGAATGGAAGAATAAAATGTAG TACG |