Optimization and validation of multi-coloured capillary electrophoresis for genotyping of Plasmodium falciparum merozoite surface proteins (msp1 and 2)
Open Access
- 23 April 2009
- journal article
- research article
- Published by Springer Science and Business Media LLC in Malaria Journal
- Vol. 8 (1), 78
- https://doi.org/10.1186/1475-2875-8-78
Abstract
Genotyping of Plasmodium falciparum based on PCR amplification of the polymorphic genes encoding the merozoite surface proteins 1 and 2 (msp1 and msp2) is well established in the field of malaria research to determine the number and types of concurrent clones in an infection. Genotyping is regarded essential in anti-malarial drug trials to define treatment outcome, by distinguishing recrudescent parasites from new infections. Because of the limitations in specificity and resolution of gel electrophoresis used for fragment analysis in most genotyping assays it became necessary to improve the methodology. An alternative technique for fragment analysis is capillary electrophoresis (CE) performed using automated DNA sequencers. Here, one of the most widely-used protocols for genotyping of P. falciparum msp1 and msp2 has been adapted to the CE technique. The protocol and optimization process as well as the potentials and limitations of the technique in molecular epidemiology studies and anti-malarial drug trials are reported.The original genotyping assay was adapted by fluorescent labeling of the msp1 and msp2 allelic type specific primers in the nested PCR and analysis of the final PCR products in a DNA sequencer. A substantial optimization of the fluorescent assay was performed. The CE method was validated using known mixtures of laboratory lines and field samples from Ghana and Tanzania, and compared to the original PCR assay with gel electrophoresis.The CE-based method showed high precision and reproducibility in determining fragment size (< 1 bp). More genotypes were detected in mixtures of laboratory lines and blood samples from malaria infected children, compared to gel electrophoresis. The capacity to distinguish recrudescent parasites from new infections in an anti-malarial drug trial was similar by both methods, resulting in the same outcome classification, however with more precise determination by CE.The improved resolution and reproducibility of CE in fragment sizing allows for comparison of alleles between separate runs and determination of allele frequencies in a population. The more detailed characterization of individual msp1 and msp2 genotypes may contribute to improved assessments in anti-malarial drug trials and to a further understanding of the molecular epidemiology of these polymorphic P. falciparum antigens.Keywords
This publication has 22 references indexed in Scilit:
- Seasonal Intermittent Preventive Treatment for the Prevention of Anaemia and Malaria in Ghanaian Children: A Randomized, Placebo Controlled TrialPLOS ONE, 2008
- A high resolution four-locus multiplex single nucleotide repeat (SNR) genotyping system in Bacillus anthracisJournal of Microbiological Methods, 2008
- Multiple-locus variable-number tandem repeat analysis of Legionella pneumophila using multi-colored capillary electrophoresisJournal of Microbiological Methods, 2008
- Comparison between agarose gel electrophoresis and capillary electrophoresis for variable numbers of tandem repeat typing of Mycobacterium tuberculosisJournal of Microbiological Methods, 2006
- Multiple-Locus Variable-Number Tandem-Repeats Analysis of Escherichia coli O157 using PCR multiplexing and multi-colored capillary electrophoresisJournal of Microbiological Methods, 2004
- Genotyping of Plasmodium falciparum infections by PCR: a comparative multicentre studyTransactions of the Royal Society of Tropical Medicine and Hygiene, 2001
- Origin of spurious multiple bands in the amplification of microsatellite sequencesMolecular Pathology, 1999
- Genetic evidence that RI chloroquine resistance of Plasmodium falciparum is caused by recrudescence of resistant parasitesTransactions of the Royal Society of Tropical Medicine and Hygiene, 1994
- Frequency of cross-fertilization in the human malaria parasitePlasmodium falciparumParasitology, 1993
- A Plasmodium falciparum MSA-2 gene apparently generated by intragenic recombination between the two allelic familiesMolecular and Biochemical Parasitology, 1991