title: An efficient PCR--SSCP-based method for detection of a chloroquine resistance marker in the PfCRT gene of Plasmodium falciparum.
abstract: The spread of chloroquine resistance throughout the world poses a major problem in combating malaria. In the present study, an efficient polymerase chain reaction-single strand conformational polymorphism (PCR--SSCP)-based assay detected the PfCRT K76T point mutation, which is a marker for chloroquine resistance. For the first time, we have used a PCR--SSCP-based technique to identify the mutation in a single-step labelling reaction during PCR and SSCP gel electrophoresis. This assay is 100% efficient, giving no false-positive or -negative results, and can be carried out within a short bench time. We have successfully analysed 120 natural isolates using the PCR-SSCP method for detection of the chloroquine resistance marker and found 91 of the 120 samples to show the PfCRT T76 mutation, and 71% (65 of the 91 samples) showed a positive correlation with chloroquine resistance from the clinical data of the patients. The PCR-SSCP technique can also be applied for the detection of new haplotypes of the PfCRT gene and surveillance of chloroquine-resistant malaria in malaria-endemic localities around the world.
authors.affiliation: Institute of Life Sciences, Nalco Square, Chandrasekharpur, Bhubaneswar 751023, India.
authors.forename: Sasmita
authors.initials: S
authors.lastname: Mishra
authors.forename: Dipak Kumar
authors.initials: DK
authors.lastname: Raj
authors.forename: R K
authors.initials: RK
authors.lastname: Hazra
authors.forename: A P
authors.initials: AP
authors.lastname: Dash
authors.forename: Prakash C
authors.initials: PC
authors.lastname: Supakar
mesh_terms: D000818:Animals; D000962:Antimalarials / Q000494:pharmacology*; D002738:Chloroquine / Q000494:pharmacology*; D016054:DNA, Protozoan / Q000302:isolation & purification; D004351:Drug Resistance / Q000235:genetics*; D005819:Genetic Markers; D006801:Humans; D007194:India; D016778:Malaria, Falciparum / Q000175:diagnosis / Q000469:parasitology; D008565:Membrane Proteins / Q000235:genetics*; D026901:Membrane Transport Proteins; D021261:Parasitic Sensitivity Tests; D010963:Plasmodium falciparum / Q000187:drug effects*; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D015800:Protozoan Proteins; D012680:Sensitivity and Specificity
chemical_list: D000962:Antimalarials; D016054:DNA, Protozoan; D005819:Genetic Markers; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C416369:PfCRT protein, Plasmodium falciparum; D015800:Protozoan Proteins; D002738:Chloroquine