Detection of Plasmodium falciparum in human blood by a nested polymerase chain reaction
Arai, M.; Mizukoshi, C.; Kubochi, F.; Kakutani, T.; Wataya, Y.
American Journal of Tropical Medicine and Hygiene 51(5): 617-626
1994
ISSN/ISBN: 0002-9637 PMID: 7985755 Document Number: 435830
A simple method for direct detection of Plasmodium falciparum parasites in infected human blood using a nested polymerase chain reaction was developed. Whole blood (10 micro l) was obtained by finger puncture and suspended in a microcentrifuge tube containing phosphate-buffered saline. For removal of components that might inhibit the PCR, blood samples were treated with saponin and washed by centrifugation. After 3 cycles of a 2-step incubation (3 minutes at 94 degrees C and 3 minutes at 55 degrees C), the first amplification was done with oligonucleotide primers specific for the junction region of the gene coding for the dihydrofolate reductase thymidylate synthase in P. falciparum. A 1- micro l portion of the first amplification was then amplified again with a second set of primers, and 226-basepair fragments were generated. The amplified products were analysed by agarose gel electrophoresis with ethidium bromide staining. Experiments with cultured parasites showed that the method could detect as few as 13 parasites in 10 micro l of whole blood. In 1991, 101 samples from 98 donors were collected in Guadalcanal, Solomon Islands. Eight of these samples gave positive results both by examination of thin blood smears and by the nested PCR. There was a correlation between parasite densities and the intensity of the results by the nested PCR. The method is suitable for detection of asymptomatic parasite carriers and evaluation of medical treatment on clinical cases.