TITLE: Amplification of P. vivax/P. malariae/P. falciparum cox3 gene in humans and non human primates
AUTHORS: Gabriela Ulloa Urizar

[DESCRIPTION]
Amplification of Plasmodium mitochondrial cytochrome c oxidase III (cox3) gene to detection Plasmodium spp. by conventional PCR. Also, detection of Plasmodium falciparum, Plasmodium vivax/simium and Plasmdium malariae/brasilianum in humans population and non human primates (NHP) by Nested-PCR.

[BEFORE_START]
Materials
Primers
Platinum II Hot Start PCR Master Mix (2X)
Nuclease-free water
Template DNA (from blood spot samples)
PCR tubes
Thermal cycler
Agarose, TAE buffer
Gel electrophoresis system
DNA stain
DNA ladder (100 bp)



To detect Plasmodium spp. in humans and non human primates use the primers:

SPP1F: 5'-CTC GCC ATT TGA TAG CGG TTA ACC-3'
SPP1R: 5'-CCT GTT ATC CCC GGC GAA CCT TC-3'

if the band you get is very faint you can do a nested PCR with the following primers:
(in my case it wasn't necessary)

SPP2F: 5'-GTA AAC ATG CWG TCA TAC ATG ATG CAC-3'
SPP2R: 5'-CCC CGG CGA ACC TTC TTA CCG T-3'

Primers for P. falciparum detection:

FALF: 5'-GAA CAC AAT TGT CTA TTC GTA CAA TTA TTC-3'
FALR: 5'-CTT CTA CCG AAT GGT TTA TAA ATT CTT TC-3'

Primers for P. vivax/P. simium detection:

VIVF: 5'-CTA GCT TTT AAC ACA ATA TTA TTG TCT ATA C-3'
VIVR: 5'-GTT CTT TTT CTA TTC AGA ATA ATG AAT ATA T-3'

Primers for P. malariae/P. brasilianum detection:

MALF: 5'-CTA GCT TTG TAC ACA AAT TAA TTC GTC TAC-3'
MALR: 5'-CTT TAT AAG AAT GAT AGA TAT TTA TGA CAT A-3'


For the PCR Mix (conventional and nested) we used the Platinum™ II Hot-Start PCR Master Mix (2X) from Invitrogen.

[GUIDELINES]
ROUND 1 PCR

A) To detect Plasmodium spp. in humans and non human primates use the primers:

1. Hydrate the primers with water without DNAases and dilute the primers for Plasmodium spp. (spp1F and spp1R) to a concentration of 2uM. 

 


2. Volumes for the pcr mix for 1 reaction (1 rx) were:

Primer spp1F [2uM]: 2 µL
Primer spp1R [2uM]: 2 µL
Master Mix (2X): 10 µL
Free water: 1 µL
Sample DNA: 5 µL

3. PCR conditions: 

96°C --- 1 min
96°C --- 10 s
63°C --- 1 min
72°C --- 1 min
The last 3 steps are repeated for 40 cycles. 
72°C --- 10 min
4°C --- store at this temperature until electrophoresis

Electrophoresis
4. Add 15 ul of PCR product to each well of the agarose gel. Then run on a 1% agarose gel prepared with TAE buffer and DNA stain at 95 volts for 30 min


 

ROUND 2 PCR 

B) To detect P. falciparum in humans and non human primates use the primers:

1.Hydrate the primers with water without DNAases and dilute the primers for P. falciparum (FALF and FALR) to a concentration of 4uM. 

 


2.Volumes for the pcr mix for 1 reaction (1 rx) were:

FALF primer [4uM]: 2 µL
FALR Primer [4uM]: 2 µL
Master Mix (2X): 10 µL
Free water: 4 µL
Diluted PCR product: 2 µL

3.PCR conditions:

96°C --- 1 min
96°C --- 10 s
54°C ---90 s
72°C --- 1 min
The last 3 steps are repeated for 30 cycles
72°C --- 10 min
4°C --- store at this temperature until electrophoresis

Electrophoresis
4. Add 15 ul of PCR product to each well of the agarose gel. Then run on a 2% agarose gel prepared with TAE buffer and DNA stain at 95 volts for35 min 

C) To detect P. vivax/P. simium in humans and non human primates use the primers:

1.Hydrate the primers with water without DNAases and dilute the primers for P. vivax (VIVF and VIVR) to a concentration of 4uM. 


 


2.Volumes for the pcr mix for 1 reaction (1 rx) were:

VIVF primer [4uM]: 2 µL
VIVR Primer [4uM]: 2 µL
Master Mix (2X): 10 µL
Free water: 4 µL
Diluted PCR product: 2 µL

3.PCR conditions:

96°C --- 1 min
96°C --- 10 s
54°C ---90 s
72°C --- 1 min
The last 3 steps are repeated for 30 cycles
72°C --- 10 min
4°C --- store at this temperature until electrophoresis


Electrophoresis
4. Add 15 ul of PCR product to each well of the agarose gel. Then run on a 2% agarose gel prepared with TAE buffer and DNA stain at 95 volts for35 min 


D) To detect P. malariae/P. brasilianum in humans and non human primates use the primers:

1.Hydrate the primers with water without DNAases and dilute the primers for P. malariae (MALF and MALR) to a concentration of 4uM. 

 


2.Volumes for the pcr mix for 1 reaction (1 rx) were:

MALF primer [4uM]: 2 µL
MALR Primer [4uM]: 2 µL
Master Mix (2X): 10 µL
Free water: 4 µL
Diluted PCR product: 2 µL

3.PCR conditions:

96°C --- 1 min
96°C --- 10 s
58°C ---90 s
72°C --- 1 min
The last 3 steps are repeated for 30 cycles
72°C --- 10 min
4°C --- store at this temperature until electrophoresis


Electrophoresis
4. Add 15 ul of PCR product to each well of the agarose gel. Then run on a 2% agarose gel prepared with TAE buffer and DNA stain at 95 volts for35 min 


[STEPS]