Abstract
Defining clone composition in Plasmodium falciparum cultures is key to verify that in vitro experiments are performed on the parasite line of interest. Genotyping of the highly polymorphic merozoite surface protein 2 gene (msp2) is a widely established method to define P. falciparum clones. Specific size variants from the two msp2 families (IC and FC27) can be used as “fingerprints” to identify individual clones in parasite mixtures. Size variant genotyping of msp2 using fluorescent nested PCR followed by fragment analysis by capillary electrophoresis (CE) provides accurate information about the presence of one or multiple parasite clones. Here, we describe an adaptation of this approach to assess the integrity and purity of P. falciparum lines kept in in vitro culture. In addition, we describe the use of synthetic mock parasite mixtures with the msp2 sequences from the parasite lines kept in culture that can provide a good estimate of the assay sensitivity, specificity, and reproducibility. We suggest that genotyping of P. falciparum lines should be performed on a regular basis as part of the standard procedures of in vitro parasite culture, as a way to secure that the parasite lines of interest are cultivated, and to monitor any cross-contamination and/or recombination events.
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Acknowledgments
We would like to thank Georges Snounou and Anne Liljander for developing the original methods as well as Manijeh Vafa Homann and Muhammad Ashgar for their contributions on the method's previous optimizations. We would also like to thank Sherwin Chan and Maria Del Pilar Quintana for their valuable comments on P. falciparum in vitro culture.
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Broumou, I., Plaza, D.F., Färnert, A. (2022). Genotyping of Plasmodium falciparum to Assess Clone Composition in Parasite Cultures. In: Jensen, A.T.R., Hviid, L. (eds) Malaria Immunology. Methods in Molecular Biology, vol 2470. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2189-9_6
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DOI: https://doi.org/10.1007/978-1-0716-2189-9_6
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