Jun 10, 2025

Public workspaceAmplification of cytb gene of Plasmodium spp. in blood spots from wild animals

DOI
https://dx.doi.org/10.17504/protocols.io.bp2l619k5vqe/v1
  • Gabriela Ulloa Urizar1
  • 1Universidade Federal Rural da Amazônia (UFRA), Belém, Pará, Brasil
Gabriela Ulloa Urizar
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.bp2l619k5vqe/v1
Collection Citation: Gabriela Ulloa Urizar 2025. Amplification of cytb gene of Plasmodium spp. in blood spots from wild animals. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l619k5vqe/v1
License: This is an open access collection distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this collection and it's working
Created: February 14, 2022
Last Modified: June 10, 2025
Collection Integer ID: 58196
Keywords: detection of the plasmodium genus, plasmodium mitochondrial, plasmodium spp, amplification of cytb gene, plasmodium genus, blood spots from wild animals amplification, cytb gene, blood spot, cytb, wild animals amplification, wildlife sample
Funders Acknowledgements:
ERANet-LAC
Grant ID: ERANet17/HLH-0271
Instituto de Salud Carlos III
Grant ID: AC18/00054
CNPq
Grant ID: 140312/2020-0
CNPq
Grant ID: 201546/2020-5
CNPq
Grant ID: 400800/2019-5
CONCYTEC
Grant ID: Nº 136-2018-FONDECYT
Abstract
Amplification of the Plasmodium mitochondrial cytochrome oxidase b gene (cytb) for detection of the Plasmodium genus in wildlife samples.
Guidelines
A) To detect Plasmodium spp. in humans and non human primates use the primers:

Procedure:
ROUND 1 PCR

1. Hydrate the primers with water without DNAases and dilute the primers for Plasmodium spp. (DW2 and DW4) to a concentration of 5uM.
Note
Final effective concentration in PCR is 0.5 µM for 20uL volume.


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

Primer DW2 [5uM]: Amount2 µL
Primer DW4 [5uM]: Amount2 µL
Master Mix (2X): Amount10 µL
Free water: Amount1 µL
Sample DNA: Amount5 µL

3. PCR conditions:

94°C --- Duration00:03:00
94°C --- Duration00:00:20
62°C --- Duration00:00:30
72°C --- Duration00:01:00
The last 3 steps are repeated for 40 cycles.
72°C --- Duration00:10:00
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 1.5% agarose gel prepared with TAE buffer at 95 volts for Duration00:40:00


Note
Before nested PCRs are performed, dilute the PCR products for Plasmodium spp. (DW2 and DW4) by 20-fold, i.e. 2ul of PCR product in 38ul of DNAase-free water. Then use this dilution as template DNA for the following nested PCR.



B) nested PCR cytb in humans and non human primates use the primers:

ROUND 2 PCR

1.Hydrate the primers with water without DNAases and dilute the primers for Plasmodium spp. (FP3 and RP3) to a concentration of 5uM.


Note
Final effective concentration in PCR is 0.5 µM for 20uL volume.


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

FP3 primer [5uM]: Amount2 µL
RP3 Primer [5uM]: Amount2 µL
Master Mix (2X): Amount10 µL
Free water: Amount4 µL
Diluted PCR product (1/20): Amount2 µL

3.PCR conditions:

94°C --- Duration00:03:00
94°C --- Duration00:00:20
52°C ---Duration00:00:30
72°C --- Duration00:01:00
The last 3 steps are repeated for 40 cycles
72°C --- Duration00:10:00
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 at 95 volts forDuration00:45:00 . Visualize bands under UV or blue light. Expected size: ~776 bp.


Materials

  • DW2 and DW4 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)
Troubleshooting
Before start
Materials
  • DW2 and DW4 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:
ROUND 1

DW2: 5'-TAA TGC CTA GAC GTA TTC CTG ATT ATC CAG-3'
DW4: 5'-TGT TTG CTT GGG AGC TGT AAT CAT AAT GTG-3'

Nested PCR cytb in humans and non human primates use the primers:
ROUND 2

FP3: 5'-TAT ATA ACT TAT TTT TTG ATA TG-3'
RP3: 5'-GTR ATW GCA TTA TCT GGA TGT GA-3'


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

Files
Protocol references
MARTINSEN, E. S., PAPERNA, I. & SCHALL, J. J. Morphological versus molecular identification of avian Haemosporidia: an exploration of three species concepts. Parasitology 133, 279–288 (2006).
Acknowledgements
This protocol has been cited in our peer-reviewed publication:
https://doi.org/10.1016/j.meegid.2024.105554

It has now been updated to include step-by-step instructions, in compliance with protocols.io formatting requirements.