Real-time PCR/MCA assay using fluorescence resonance energy transfer for the genotyping of resistance related DHPS-540 mutations in Plasmodium falciparum

Hdl Handle:
http://hdl.handle.net/10144/116333
Title:
Real-time PCR/MCA assay using fluorescence resonance energy transfer for the genotyping of resistance related DHPS-540 mutations in Plasmodium falciparum
Authors:
Mens, Petra F; van Overmeir, Chantal; Bonnet, Maryline; Dujardin, Jean-Claude; d'Alessandro, Umberto
Journal:
Malaria Journal
Abstract:
BACKGROUND: Sulphadoxine-pyrimethamine has been abandoned as first- or second-line treatment by most African malaria endemic countries in favour of artemisinin-based combination treatments, but the drug is still used as intermittent preventive treatment during pregnancy. However, resistance to sulphadoxine-pyrimethamine has been increasing in the past few years and, although the link between molecular markers and treatment failure has not been firmly established, at least for pregnant women, it is important to monitor such markers. METHODS: This paper reports a novel sensitive, semi-quantitative and specific real-time PCR and melting curve analysis (MCA) assay using fluorescence resonance energy transfer (FRET) for the detection of DHPS-540, an important predictor for SP resistance. FRET/MCA was evaluated using 78 clinical samples from malaria patients and compared to PCR-RFLP. RESULTS: Sixty-two samples were in perfect agreement between both assays. One sample showed a small wild type signal with FRET/MCA that indicates a polyclonal infection. Four samples were not able to generate enough material in both assays to distinguish mutant from wild-type infection, six samples gave no signal in PCR-RFLP and five samples gave no amplification in FRET/MCA. CONCLUSION: FRET/MCA is an effective tool for the identification of SNPs in drug studies and epidemiological surveys on resistance markers in general and DHPS-540 mutation in particular.
Affiliation:
Prince Leopold Institute of Tropical Medicine, Department of Parasitology, Antwerp, Belgium; Koninklijk Instituut voor de Tropen/Royal Tropical Institute, KIT Biomedical Research, Amsterdam, The Netherlands; Epicentre, Paris, France
Issue Date:
17-Mar-2008
URI:
http://hdl.handle.net/10144/116333
DOI:
10.1186/1475-2875-7-48
PubMed ID:
18346279
Additional Links:
http://www.malariajournal.com/content/7/1/48
Type:
Article
Language:
en
ISSN:
1475-2875
Appears in Collections:
Malaria

Full metadata record

DC FieldValue Language
dc.contributor.authorMens, Petra Fen
dc.contributor.authorvan Overmeir, Chantalen
dc.contributor.authorBonnet, Marylineen
dc.contributor.authorDujardin, Jean-Claudeen
dc.contributor.authord'Alessandro, Umbertoen
dc.date.accessioned2010-11-25T16:33:23Z-
dc.date.available2010-11-25T16:33:23Z-
dc.date.issued2008-03-17-
dc.identifier.citationMalar. J. 2008;7:48en
dc.identifier.issn1475-2875-
dc.identifier.pmid18346279-
dc.identifier.doi10.1186/1475-2875-7-48-
dc.identifier.urihttp://hdl.handle.net/10144/116333-
dc.description.abstractBACKGROUND: Sulphadoxine-pyrimethamine has been abandoned as first- or second-line treatment by most African malaria endemic countries in favour of artemisinin-based combination treatments, but the drug is still used as intermittent preventive treatment during pregnancy. However, resistance to sulphadoxine-pyrimethamine has been increasing in the past few years and, although the link between molecular markers and treatment failure has not been firmly established, at least for pregnant women, it is important to monitor such markers. METHODS: This paper reports a novel sensitive, semi-quantitative and specific real-time PCR and melting curve analysis (MCA) assay using fluorescence resonance energy transfer (FRET) for the detection of DHPS-540, an important predictor for SP resistance. FRET/MCA was evaluated using 78 clinical samples from malaria patients and compared to PCR-RFLP. RESULTS: Sixty-two samples were in perfect agreement between both assays. One sample showed a small wild type signal with FRET/MCA that indicates a polyclonal infection. Four samples were not able to generate enough material in both assays to distinguish mutant from wild-type infection, six samples gave no signal in PCR-RFLP and five samples gave no amplification in FRET/MCA. CONCLUSION: FRET/MCA is an effective tool for the identification of SNPs in drug studies and epidemiological surveys on resistance markers in general and DHPS-540 mutation in particular.en
dc.language.isoenen
dc.relation.urlhttp://www.malariajournal.com/content/7/1/48en
dc.rightsPublished by BioMed Central, [url]http://www.malariajournal.com/[/url] Archived on this site by Open Access permissionen
dc.subject.meshAnimalsen
dc.subject.meshDemocratic Republic of the Congoen
dc.subject.meshDihydropteroate Synthaseen
dc.subject.meshDrug Resistanceen
dc.subject.meshFluorescence Resonance Energy Transferen
dc.subject.meshHumansen
dc.subject.meshMalaria, Falciparumen
dc.subject.meshMutationen
dc.subject.meshPlasmodium falciparumen
dc.subject.meshPolymerase Chain Reactionen
dc.subject.meshPolymorphism, Single Nucleotideen
dc.subject.meshSensitivity and Specificityen
dc.subject.meshTransition Temperatureen
dc.titleReal-time PCR/MCA assay using fluorescence resonance energy transfer for the genotyping of resistance related DHPS-540 mutations in Plasmodium falciparumen
dc.typeArticleen
dc.contributor.departmentPrince Leopold Institute of Tropical Medicine, Department of Parasitology, Antwerp, Belgium; Koninklijk Instituut voor de Tropen/Royal Tropical Institute, KIT Biomedical Research, Amsterdam, The Netherlands; Epicentre, Paris, Franceen
dc.identifier.journalMalaria Journalen

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