Skip to main content

Table 1 Primers sequences and real-time PCR conditions used to detect Plasmodium species, Cambodia, 2011

From: Performance of “VIKIA Malaria Ag Pf/Pan” (IMACCESS®), a new malaria rapid diagnostic test for detection of symptomatic malaria infections

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