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Twelve microsatellite markers for characterization of Plasmodium falciparum from finger-prick blood samples

Published online by Cambridge University Press:  01 August 1999

T. J. C. ANDERSON ,
XIN-ZHUAN SU ,
M. BOCKARIE ,
M. LAGOG  and
K. P. DAY
T. J. C. ANDERSON
Affiliation:
Wellcome Trust Centre for the Epidemiology of Infectious Disease, Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK
XIN-ZHUAN SU
Affiliation:
Laboratory of Parasitic Diseases, NIAID, NIH, Rm 126, Building 4, 9000 Rockville Pike, Bethesda, MD 20892, USA
M. BOCKARIE
Affiliation:
Papua New Guinea Institute for Medical Research, PO Box 378, Madang, Papua New Guinea
M. LAGOG
Affiliation:
Papua New Guinea Institute for Medical Research, PO Box 378, Madang, Papua New Guinea
K. P. DAY
Affiliation:
Wellcome Trust Centre for the Epidemiology of Infectious Disease, Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, UK
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Abstract

Multiple, selectively neutral genetic markers are the most appropriate tools for analysis of parasite population structure and epidemiology, but yet existing methods for characterization of malaria field samples utilize a limited number of antigen encoding genes, which appear to be under strong selection. We describe protocols for characterization of 12 microsatellite markers from finger-prick blood samples infected with Plasmodium falciparum. A two-step, heminested strategy was used to amplify all loci, and products were visualized by fluorescent end-labelling of internal primers. This procedure allows amplification from low levels of template, while eliminating the problem of spurious products due to primer carry over from the primary round of PCR. The loci can be conveniently multiplexed, while accurate sizing and quantification of PCR products can be automated using the GENOTYPER software. The primers do not amplify co-infecting malaria species such as P. vivax and P. malariae. To demonstrate the utility of these markers, we characterized 57 infected finger-prick blood samples from the village of Mebat in Papua New Guinea for all 12 loci, and all samples were genotyped a second time to measure reproducibility. Numbers of alleles per locus range from 4 to 10 in this population, while heterozygosities range from 0·21 to 0·87. Reproducibility (measured as concordance between predominant alleles detected in replicate samples) ranged from 92 to 98% for the 12 loci. The composition of PCR products from infections containing multiple malaria clones could also be defined using strict criteria and scored in a highly repeatable manner.

Keywords

microsatellitePlasmodium falciparumheterozygosity
Type
Research Article
Information
Parasitology , Volume 119 , Issue 2 , August 1999 , pp. 113 - 125
Copyright
1999 Cambridge University Press

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