Multiplex qPCR for Detection and Absolute Quantification of Malaria
Open accessAbstract
We describe development of an absolute multiplex quantitative real-time PCR for detection of Plasmodium spp., P. falciparum and P. vivax targets in order to produce an assay amenable to high throughput but with reduced costs. Important qPCR experimental details and information that is critical to performance and reliability of assay results were investigated. Inhibition studies were performed to test and compare co-purification of PCR inhibitors in samples extracted from whole blood using either the manual or automated methods. To establish the most optimal qPCR reaction volume, volume titration of the reaction master mix was performed starting at 10 µl to 1 µl reaction master mix with 1 µl of template DNA in each reaction. As the reaction volume decreased, qPCR assays became more efficient with 1 µl reaction master mix being the most efficient. For more accurate quantification of parasites in a sample, we developed plasmid DNAs for all the three assay targets for absolute quantification. All of absolute qPCR assays performed with efficiency of more than 94%, R(2) values greater than 0.99 and the STDEV of each replicate was <0.167. Linear regression plots generated from absolute qPCR assays were used to estimate the corresponding parasite density from relative qPCR in terms of parasite/µl. One copy of plasmid DNA was established to be equivalent to 0.1 parasite/µl for Plasmodium spp. assay, 0.281 parasites for P. falciparum assay and 0.127 parasite/µl for P. vivax assay. This study demonstrates for the first time use of plasmid DNA in absolute quantification of malaria parasite. The use of plasmid DNA standard in quantification of malaria parasite will be critical as efforts are underway to harmonize molecular assays used in diagnosis of malaria.
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A seasoned microscopist can discern parasitemias only above approximately 100 parasites per microliter, whereas PCR can detect 1 parasite per microliter or less.
“A seasoned microscopist is only able to discern parasitemias above a density of approximately 100 parasites per microliter 6,7 compared with PCR where detection can occur at 1 parasite per microliter or less. 8”
From What is the burden of submicroscopic malaria in pregnancy in central India? (2015) · cites this paper as [8]
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What is the burden of submicroscopic malaria in pregnancy in central India?
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- Authors
- Kamau, E., Alemayehu, S., Feghali, K. C., Saunders, D., & Ockenhouse, C. F.
- Affiliations
- Walter Reed Army Institute of Research (US)
- Armed Forces Research Institute of Medical Science (TH)
- Corresponding author
- Kamau, Edwin
- Venue
- PLoS ONE
- Publisher
- Public Library of Science (PLoS)
- Published
- 2013-08-29
- Volume
- 8
- Issue
- 8
- Pages
- e71539
- ISSN
- 1932-6203 (ISSN-L)
- PMC
- PMC3756973
- OpenAlex
- W2010146342
- Cited by
- 156
- References
- 20
- Citations by year
- 2026: 13 · 2025: 7 · 2024: 15 · 2023: 13 · 2022: 11 · 2021: 19 · 2020: 20 · 2019: 18 · 2018: 8 · 2017: 7 · 2016: 5 · 2015: 10 · 2014: 9
- FWCI
- 8.18
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- PLoS ONE (publishedVersion, public-domain)
- Europe PMC (PubMed Central) (publishedVersion, other-oa)
- PubMed Central (publishedVersion, public-domain)
- PubMed (publishedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Greater South Information System
- Funders
- U.S. Department of Defense
- Topics
- Health Sciences › Medicine › Public Health, Environmental and Occupational Health › Malaria Research and Control
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Physical Sciences › Engineering › Biomedical Engineering › Biosensors and Analytical Detection
- Keywords
- malaria, Plasmodium falciparum, Plasmodium vivax, multiplex real-time PCR, absolute quantification, parasite density, PCR inhibition, malaria diagnosis
- MeSH terms
- Humans, Malaria, Plasmodium, Plasmodium falciparum, Plasmodium vivax, Sensitivity and Specificity, Reproducibility of Results, Parasite Load, Multiplex Polymerase Chain Reaction, Real-Time Polymerase Chain Reaction
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