Apr 08, 2026
One-Step Genotyping Protocol for Detection of Plasmodium falciparum hrp2 Gene Deletions
- 1International Center for Research and Training on Applied Genomics and Health Surveillance (CIGASS) Cheikh Anta Diop University (UCAD)
Protocol Citation: Djiby SOW, Awa Deme 2026. One-Step Genotyping Protocol for Detection of Plasmodium falciparum hrp2 Gene Deletions. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg3226zv25/v1
License: This is an open access protocol 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 protocol and it's working
Created: May 30, 2024
Last Modified: April 08, 2026
Protocol Integer ID: 100892
Keywords: plasmodium falciparum hrp2 gene deletion, significant challenge to malaria diagnosis, malaria diagnosis, malaria research, emergence of plasmodium, hrp2 antigen detection, plasmodium, hrp2 gene deletion, accurate detection of hrp2, hrp2 gene, guiding diagnostic strategy, parasites with deletion, antigen detection, deleted parasite, step pcr, diagnostic strategy, genotyping approach, rapid diagnostic test, assay validity, parasite, pcr, molecular biology, endemic setting, laboratory workflow, approach for the detection, assay
Disclaimer
This protocol is provided for research and surveillance purposes only and is not intended for clinical diagnosis or patient management. The authors make no warranties, express or implied, regarding the accuracy, completeness, or performance of this method. Users are responsible for ensuring that the protocol is applied in accordance with applicable institutional guidelines, regulatory requirements, and biosafety standards.
The authors shall not be held liable for any direct or indirect damages resulting from the use or misuse of this protocol. It is the responsibility of the user to validate the method within their specific laboratory setting before implementation.
Abstract
The emergence of Plasmodium falciparum parasites with deletions in the hrp2 gene poses a significant challenge to malaria diagnosis, particularly for rapid diagnostic tests (RDTs) that rely on HRP2 antigen detection. Reliable and scalable methods are therefore essential for monitoring the prevalence and distribution of these deletions.
This protocol describes a rapid and simplified one-step PCR-based genotyping approach for the detection of hrp2 gene deletions in P. falciparum. The method is designed to streamline laboratory workflows by reducing processing time while maintaining high sensitivity and specificity. It incorporates appropriate positive, negative, and blank controls to ensure assay validity and reproducibility.
Intended for use by trained personnel with a background in molecular biology, this protocol supports malaria research and surveillance activities. It is particularly suitable for implementation in endemic settings where timely and accurate detection of hrp2-deleted parasites is critical for guiding diagnostic strategies and public health interventions.
Purpose
This protocol provides a method for performing Plasmodium falciparum histidine-rich protein-2 (PfHRP2) genotyping on genomic DNA extracted from either dried blood spots (DBSs) on Whatman 903 filter paper or whole blood.
Scope
This SOP applies to all clinical research and surveillance activities related to Plasmodium falciparum malaria. It is intended for use by technical personnel with a background in molecular biology and is strictly limited to research and surveillance purposes.
Reagents and Materials
1.0 Equipment/Materials (if applicable)
| A | B | |
| Item | Definition | |
| Multichannel Pipettes | Rainin LTS Pipet-Lite Multichannel series (or equivalent) | |
| Single channel Pipettes | Rainin LTS Pipet-Lite XLS Single channel series (or equivalent) | |
| Centrifuge | Bench top centrifuge with 96-well microwell plate rotor e.g. Sorvall Legend RT (or equivalent) | |
| Thermocycler | BioRad C1000 Thermal Cycler (or equivalent) | |
| 4°C Fridge | Thermo Scientific Revco (or equivalent) | |
| -20°C Freezer | Kenmore (or equivalent) |
1.0 Reagents and Media
1.1
Reagents:
| A | B | C | |
| Material Name | Other Information | Storage Condition | |
| Q5 5x Buffer | -10°C to -30°C | ||
| Q5 High-fid enzyme | -10°C to -30°C | ||
| dNTP Mix (10mM each) | 10mM each | -10°C to -30°C | |
| Primers | Forward and Reverse | -10°C to -30°C | |
| Nuclease Free Water | Ambient Temperature |
1.1 Primers:
| A | B | C | D | |
| Gene name | Primer name | Primer direction | Primer sequence | |
| pfhrp2 | Bravo_f1 | Forward | 5’- ATGATTCATTATTCTATATTTATAAGGAAGATTAC-3’ | |
| Bravo_r1 | reverse | 5’- CACTTCATGTATTTATGTATGCAGAAC- 3’ |
8.0 Supplies, Other Materials
1.1 96-well PCR plate, half-skirt, stored at ambient temperature
1.2 MicroAmp Clear Adhesive Film, stored at ambient temperature
1.3 PCR workstations with UV light
1.4 Vortex
1.5 1.5 or 2.0 ml sterile RNAse and DNAse-free tubes
1.6 Tube racks
1.7 DNAse-free or sterile solution reservoirs or basins 1.8 Benchtop cooler or ice box
1.9 Microwave for agarose gel preparation
1.10 Standard top-load balance
1.11 Agarose gel electrophoresis apparatus and combs
1.12 Power supply for gel systems
1.13 Bioimage system or UV transilluminator
1.14 Biohazard bag with holder or plastic container for tip and tube disposal
1.15 Powder-free latex, vinyl or nitrile gloves
1.16 Kimwipes
1.17 Permanent markers
1.18 Clean laboratory coat and protective goggles
1.19 70% ethanol in spray bottle
9.0 Safety Precautions
1.1 Wear personal protective equipment (PPE) at a minimum a lab coat, gloves and
goggles.
1.2 Adhere to established biosafety guidelines and follow recognized standards outlined in microbiological and biomedical laboratory safety manuals.
1.3 Universal precautions for working with infectious agents must be followed when performing
this assay.
1.4 PCR cabins and crosslinker are equipped with UV lights, cautions should be considered during decontamination.
1.5 Use aerosol-resistant pipette tips to minimize PCR contamination.
1.6 Decontaminate all surfaces and pipettes with 70% ethanol, before and after each use according
to DPDM.DR.C.003, Decontamination of Work Area and Waste Material and use of Disinfectant.
1.7 Follow safety data sheets (SDS) when handling all chemicals including those known to
be carcinogenic and reproductively toxic.
11.0 Quality Control
1.1 Positive and negative Controls
1.1.1. The positive controls are obtained from cultured P. falciparum strains, such as 3D7 or 7G8 (wild type), Dd2 (mutant-pfhrp2 deleted) stored in a laboratory
freezer set to -20°C, or in a laboratory freezer set to -80°C for long-term storage.
1.1.2. Negative control is normal human DNA, stored in a laboratory freezer set to -20°C.
1.1.3. Blank control well without DNA template must be run with samples in the same plate.
1.1.4. Repeat run if control results are invalid. (See Section 11.2)
1.2 When required, the amplified DNA
products will be confirmed by gel electrophoresis. PCR will be repeated if:
1.2.1. The major product is not the expected size.
1.2.2. The major product is of the correct size, but too much smearing exists.
1.2.3. There is no PCR product visible. NOTE: a negative result will occur for pfhrp2-deleted samples. Following an initial negative pfhrp2 PCR result,
the PCR should be repeated up to two more times. If negative for all 3
reactions, WHO guidelines need to be followed to complete identification of pfhrp2-deletion.
1.2.4. The negative control has a product band.
1.2.5. If re-amplification is not valid, DNA re-extraction should be performed at least
once.
PROTOCOL
Workflow or Process Table N/A : Examination (Test) Procedure
1 Amplification of PfHRP2 gene fragment
1. To prevent contamination with amplicons, preparation of primers and master mix buffer must be performed in different and/or physically separated work areas. Follow the recommendations of the lab.
2. Turn on the UV light in the PCR Workstation for 20 minutes (or as recommended by the manufacturer) before use.
3. PCR master mix preparation should be performed on your lab bench space (unless otherwise advised).
4. Thaw PCR reagents (dNTP mix, primers, and Q5 Buffer) at room temperature except for enzymes. Vortex and microcentrifuge briefly and place in bench top cooler.
5. Remove enzyme from -20ºC freezer and leave on ice or bench top cooler.
7. Prepare the master mix based on the following amounts of reagents for a 50µl PCR reaction in 1.5 ml tube or solution basin.
| A | B | |
| Primary | ||
| Reagents | µL/Sample | |
| Q5 5x buffer (NEB) | 10 | |
| dNTP mix (10mM) | 1 | |
| Bravo_f1 (10µM) | 0.63 | |
| Bravo_r1 (10µM) | 0.63 | |
| Q5 High Fidelity Enzyme Mix | 0.5 | |
| H20 | 32.24 | |
| Template DNA (add to tube separately) | 5 | |
| Final reaction volume | 50 |
8. Mix and place the master tube or basin on ice.
9. Label 200µl thin-walled PCR tubes or label a 96-well plate (according to testing format).
10. Add 45µl of the master mix into each PCR tube or well of PCR plate.
11. Take the PCR tubes (or plates) with the mastermix to the PCR Workstation and add 5µl isolated DNA template into the corresponding tube or wells.
12. Change pipette tip between each sample each time.
13. Cap the tube or seal the PCR plate tightly.
14. Briefly centrifuge (5-10 seconds) to collect all the reagents into the bottom of the tube. Bring the tubes or plate to PCR machine room. Load tubes or plate into the thermocycler machine.
15. Select pre-programmed PCR protocol or set up a new protocol based on the cycling profiles in the table below.
16. Check the accuracy of the program before pressing the run button. Only transfer to the thermocycler once 98°C has been reached.
| A | B | |
| One Cycle (hold) | ||
| 98°C | 3 minutes | |
| 30 PCR cycles | ||
| 98°C | 30 seconds | |
| 60°C | 90 seconds | |
| 68°C | 2 minutes | |
| One cycle (hold) | ||
| 68°C | 5 minutes | |
| 4°C | ∞ |
17. Proceed immediately to the Agarose gel preparation or place the PCR plate in the 4℃ refrigerator overnight.
2 Agarose gel preparation to confirm the PfHRP2 amplification results Note: All the gel electrophoresis steps should be performed in the gel room
1. Prepare 1% (w/v) agarose solution. Add 1.0 g agarose and 100 ml 1x TBE to a glass flask or bottle with a magnetic spin bar.
2. Carefully microwave agarose gel solution until boiling (1 to 2 minutes at full power). Swirl using heat resistant glove to prevent heat injury. Repeat until agarose has completely dissolved into solution.
3. Cool solution to 60°C on a magnetic plate spinner.
4. Add µl of GelRed (10,000x stock) to the 100 ml agarose solution for a 1x solution. Mix thoroughly.
5. Set up gel-casting tray and comb and carefully pour agarose gel solution into the gel cast.
6. Remove any bubbles and allow agarose to solidify at room temperature (approximately 30 minutes).
7. Once agarose has solidified, gently remove the comb(s).
8. Place gel into electrophoresis apparatus with sample wells closest to the negative (black) end of the gel chamber.
9. Slowly pour 1x TBE running buffer to the fill line of gel-running apparatus and ensure that the gel is completely submerged in the buffer.
10. Add 2µl of 5x DNA loading dye into a new tube or on the surface of a parafilm strip for each sample to be run.
11. Add 8µl of PCR product to the loaded 5x dye and mix well.
12. Load 8µl of the appropriate molecular marker in the first well
13. Load 8µl of each sample to the wells; change pipette tip with each sample to prevent cross-contamination.
14. Run the gel at 100V for about 1 hour to migrate gel loading dye front and separate the DNA samples.
15. Check to see that loaded material has run toward the positive (red) end of the gel box sufficiently to visualize the bands and identify specific sizes.
16. Utilize the Bioimage system or UV transilluminator to produce an image of the gel for recording.
17. The expected secondary PCR band size is 1300 bp for in-tact pfhrp2. Please note, this size can vary in the instance of partial deletion.
References
Jones S, Subramaniam G, Plucinski MM, Patel D, Padilla J, Aidoo M, Talundzic E. One-step PCR: A novel protocol for determination of pfhrp2 deletion status in Plasmodium falciparum. PLoS One. 2020 Jul 23;15(7):e0236369. doi: 10.1371/journal.pone.0236369. PMID: 32702040; PMCID: PMC7377462.