title: Colorimetric detection of Plasmodium falciparum and direct sequencing of amplified gene fragments using a solid phase method.
abstract: A rapid colorimetric assay for the detection of DNA from Plasmodium falciparum malaria is described, allowing direct sequencing of amplified fragments in the positive samples. The method is based on amplification by the polymerase chain reaction (PCR), with incorporation of biotin and a lac operator sequence in the amplified target DNA. The PCR product was immobilized on streptavidin-coupled magnetic beads, and detected by the specific binding of an Escherichia coli lac repressor beta-galactosidase fusion protein. Positive samples were subsequently treated with alkali to generate single stranded templates, which were used for solid phase genomic sequencing. As targets for amplification and sequencing we selected a region of the gene for the antigen Pf155/RESA and a region of the parasite dihydrofolate reductase gene (PfDHFR/TS). We show here that both of these gene targets can be used for specific detection of P. falciparum in patient blood samples. Genomic sequencing of five patient isolates revealed no variation in the Pf155/RESA gene fragment. In a comparison of this sequence with conserved protein domains, a marked similarity to the src homology region 3 was detected. A point mutation was found in the PfDHFR/TS gene fragment of one of the clinical samples, replacing Ser108 with Asn. This mutation has earlier been described in pyrimethamine and cycloguanile-resistant strains of P. falciparum.
authors.affiliation: Department of Medical Genetics, Biomedical Center, Uppsala, Sweden.
authors.forename: M
authors.initials: M
authors.lastname: Holmberg
authors.forename: J
authors.initials: J
authors.lastname: Wahlberg
authors.forename: J
authors.initials: J
authors.lastname: Lundeberg
authors.forename: U
authors.initials: U
authors.lastname: Pettersson
authors.forename: M
authors.initials: M
authors.lastname: Uhlén
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000235:genetics; D001483:Base Sequence; D001710:Biotin; D003124:Colorimetry / Q000379:methods*; D004252:DNA Mutational Analysis; D015342:DNA Probes; D016054:DNA, Protozoan / Q000032:analysis*; D004277:DNA, Single-Stranded; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D006801:Humans; D007763:Lac Operon; D016778:Malaria, Falciparum / Q000097:blood / Q000469:parasitology*; D008863:Microspheres; D008969:Molecular Sequence Data; D010963:Plasmodium falciparum / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D011519:Proto-Oncogenes; D015800:Protozoan Proteins / Q000235:genetics; D016415:Sequence Alignment; D017385:Sequence Homology; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics
chemical_list: D000953:Antigens, Protozoan; D015342:DNA Probes; D016054:DNA, Protozoan; D004277:DNA, Single-Stranded; D015800:Protozoan Proteins; D001710:Biotin; D013762:Tetrahydrofolate Dehydrogenase