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.