Section 4
Chapter 3,092

Development and comparison of quantitative assays for the dihydropteroate synthetase codon 540 mutation associated with sulfadoxine resistance in Plasmodium falciparum

Shaio, M.F.; Wang, P.; Lee, C.S.; Sims, P.F.; Hyde, J.E.

Parasitology 116: 203-210


ISSN/ISBN: 0031-1820
PMID: 9550212
DOI: 10.1017/s0031182097002187
Accession: 003091366

A point mutation in codon 540 of the dihydropteroate synthetase (dhps) gene affecting sulfadoxine resistance has previously been found in parasites from patients with Plasmodium falciparum infection. Here, we investigated 4 methods of identifying this mutation in clinical specimens and established a reliable quantitative assay to estimate the percentage of resistant type in mixed infections. A diagnostic PCR assay based on allele-specific amplification was developed, which clearly typed the clinical specimens examined. The mutation in codon 540 introduces an additional FokI cleavage site which provided a second method to differentiate mutant from wild type, where the former gives rise to 2 characteristic fragments of 538 and 326 bp that are absent from the latter. To calibrate quantitatively the ratio of alleles in mixed samples, we constructed artificial mixes containing 2 plasmid DNAs, one carrying the mutation and the other a wild-type insert. When 32P-labelling was employed, the allele-specific PCR assay could detect the level of resistant type in a mixture down to 0.1-1%, while for the restriction enzyme/PCR analysis, the figure was approximately 10%. Furthermore, neither fluorescent dye-labelled terminator nor dye-labelled primer cycle sequencing was able to detect the mutant allele if it was present at less than 20-30%. We conclude that the allele-specific PCR assay is the most sensitive method of detecting the codon 540 mutation in P. falciparum dhps, and the method of choice for estimating the composition of mixed samples.

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