Plasmodium falciparum: a simple polymerase chain reaction method for differentiating strains.
Laboratory studies of the protozoan parasite Plasmodium falciparum have been hampered by difficulties in defining differences between isolates. We have developed a method based on the polymerase chain reaction (PCR) that allowed us to identify quickly the various strains with which we routinely work. We also adapted methods for easily purifying enough DNA to produce a PCR product from a small volume of culture: 100 microliters of an in vitro culture infected at 1% parasitemia. The primers were chosen from conserved regions flanking the variable repeats in four cloned genes, RESA, MSA-1, MSA-2, and CSP. The PCR products amplified from three of these genes differed in size and allowed us to identify particular isolates on this basis alone. The variation was between strains, and not a reflection of genetic instability during in vitro culture of one isolate. The method is sufficiently sensitive to detect a 1% contamination of one strain with another, an advantage for monitoring the integrity of strains when different isolates are grown in the same laboratory. The technical ease and speed of this assay and the small amount of culture required make it ideal for monitoring strains in the laboratory.
Wooden
J
J
Department of Genetics (SK-50), University of Washington, Seattle 98195.
Gould
E E
EE
Paull
A T
AT
Sibley
C H
CH
D000818:Animals; D000953:Antigens, Protozoan / Q000235:genetics; D000954:Antigens, Surface / Q000235:genetics; D001483:Base Sequence; D016054:DNA, Protozoan / Q000235:genetics; D020066:Merozoite Surface Protein 1; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D010963:Plasmodium falciparum / Q000145:classification* / Q000235:genetics; D016133:Polymerase Chain Reaction; D011498:Protein Precursors / Q000235:genetics; D015800:Protozoan Proteins / Q000235:genetics
D000953:Antigens, Protozoan; D000954:Antigens, Surface; D016054:DNA, Protozoan; D020066:Merozoite Surface Protein 1; D009838:Oligodeoxyribonucleotides; D011498:Protein Precursors; D015800:Protozoan Proteins; C047712:S-antigen, Plasmodium falciparum; C041595:circumsporozoite protein, Protozoan; C050256:ring-infected erythrocyte surface antigen (RESA), Plasmodium falciparum