Detection of Plasmodium falciparum in human blood by a nested polymerase chain reaction.
We have developed a simple method for direct detection of Plasmodium falciparum parasites in infected human blood using a nested polymerase chain reaction. Whole blood (10 microliters) 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 three cycles of a two-step incubation (3 min at 94 degrees C and 3 min 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-microliter 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 analyzed 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 microliters of whole blood. In 1991, 101 samples from 98 donors were collected in Guadalcanal, Solomon Islands. Eight of these samples gave positive results by both 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.
Arai
M
M
Faculty of Pharmaceutical Sciences, Okayama University, Japan.
Mizukoshi
C
C
Kubochi
F
F
Kakutani
T
T
Wataya
Y
Y
D000293:Adolescent; D000328:Adult; D000818:Animals; D000962:Antimalarials / Q000627:therapeutic use; D001483:Base Sequence; D002648:Child; D017931:DNA Primers / Q000737:chemistry; D016054:DNA, Protozoan / Q000737:chemistry; D003720:Densitometry; D004311:Double-Blind Method; D004587:Electrophoresis, Agar Gel; D005260:Female; D006801:Humans; D016778:Malaria, Falciparum / Q000175:diagnosis* / Q000188:drug therapy; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D018512:Parasitemia / Q000175:diagnosis* / Q000188:drug therapy; D010963:Plasmodium falciparum / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D013940:Thymidylate Synthase / Q000235:genetics
D000962:Antimalarials; D017931:DNA Primers; D016054:DNA, Protozoan; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase