Plasmodium falciparum: Detection of Antifolate Resistance by Mutation-Specific Restriction Enzyme Digestion

Point mutations in dihydrofolate reductase and dihydropteroate synthase genes of Plasmodium falciparum isolates from Venezuela.

“…The parasite genomic DNA was extracted from infected red blood cells using proteinase K, followed by extraction with phenol-chloroform and ethanol precipitation. Analysis of mutations at the DHFR 16 and 108 sites was done using the PCR method followed by restriction enzyme digestion as previously described, 17 with the following modifications: 50-100 ng of genomic DNA, 1 M of each primer, 200 M of each DNTP, buffer (50 mM KCl, 10 mM Tris, pH 8.4, 1.5 mM MgCl 2 , 0.1 mg/ml of gelatin), and 2.5 units of Taq polymerase were used in a 100-l reaction. The denaturation was performed at 94ЊC for 5 min, the samples were then incubated at 94ЊC for 45 sec, 50ЊC for 1 min, 72ЊC for 2 min for 40 cycles, and a final extension was done at 72ЊC for 3 min.…”

“…The enzyme Nla III was used to digest Ala-16 (wild) or Val-16 (mutant), showing a pattern characteristic for each one as described. 17 Digestions were done in 40-l reactions containing 10 l of purified PCR fragments according to the manufacturer's (New England Biolabs. Inc., Beverly, MA) specifications.…”

“…The codon sites 50, 51, 59, and 164 in the DHFR gene were studied using methods previously described. [9][10][11][12][13][14][15][16][17][18]19 The DHFR domain was first amplified in a primary round of PCR using the primers AMPI/AMP2 as described. 18 An aliquot of 0.1-0.5 l of the first amplification was used in a mutation-specific second round PCR to detect the DHFR mutation sites 51, 59, and 164, as described.…”

“…18 An aliquot of 0.1-0.5 l of the first amplification was used in a mutation-specific second round PCR to detect the DHFR mutation sites 51, 59, and 164, as described. [9][10][11][12][13][14][15][16][17][18] For the analysis of the DHFR 50 codon, we used mutationspecific sense primers FR50w (5Ј-GGAGTATTACCA-TGGAAAT-3Ј), which specifically amplifies the sequence containing the wild type TGT (Cys) codon, and FR50m (5Ј-GGAGTATTACCATGGAAAC-3Ј), which is specific for the mutant codon CGT (Arg); these primers were paired with the common antisense primer SP2 19 to give a final product of 570 basepairs. The cycling parameters for second round were initial denaturation at 95ЊC for 3 min, followed by 15 cycles of denaturation at 92ЊC for 30 sec, annealing at 55ЊC for 45 sec, and extension at 72ЊC for 45 sec, and a final extension at 72ЊC for 3 min.…”

Plasmodium falciparum: selection of serine 108 of dihydrofolate reductase during treatment of uncomplicated malaria with co-trimoxazole in Ugandan children.

“…12 Fortyone children with symptomatic malaria from the Kabarole and Bundibugyo Districts of western Uganda were recruited for the study. Data were obtained about polymorphisms in the DHFR and DHPS genes that have been associated with antifolate resistance [5][6][7][13][14][15][16][17][18][19] by extraction and amplification of parasite DNA from filter paper and thick blood films. We report on a comparison of the prevalence of polymorphisms of the antifolate target genes at days 0 and 3/7 and their association with in vivo results in one subsample of these children.…”