Assessment of a Synthetic DNA Probe for Plasmodium falciparum in African Blood Specimens

Synthetic DNA oligomers homologous to 21-base long repetitive sequences ofPlasmodiumfakiparum DNA were labeled with 32Pusing T4 kinase, and were hybridized with purified DNA and with processed blood samples from Africa. The sequence PFR1, its antiparallel oligomer PFR1R, and PFR1 covalently attached to biotin hybridized sim ilarly to P.fakiparum DNA. One-microliter aliquots of blood from Zaire spotted on prewet nylon filters and hybridized with PFR1 gave detectable autoradiogram signals from samples with parasitemias as low as 1,000 parasites/mm3. Blood lysis and protein digestion followed by alkylation allowed dot-blot processing of larger aliquots of blood. After hybridization with PFR 1 and autoradiography, 26 samples were scored positive visually, compared with 34 scored positive by microscopy. The effective sensitivity for processed l0-@l samples was about 500 parasites/mm3. Signals from hybridized probes were quantitated by liquid scintillation counting and densitometry, and were proportional to the amounts of purified P. fakiparum DNA applied to the filter. Autoradiogram signals also were roughly pro portional (correlation coefficient, r = 0.77) to the number of parasites/mm3 of blood from field samples as determined by microscopic examination. Because of the existence of highly repetitive, species-specific DNA in eukaryotic organisms,' it is anticipated that DNA-based diagnostic probes can be useful diagnostic reagents for para sitic infections. Malaria, Chagas' disease, leish maniasis, and babesiosis are four diseases caused by protozoa for which DNA probes representing repetitive, species-specific sequences have been described.25 The detection of hemoparasites is made somewhat easier because blood contains a relatively low density of host DNA compared to other tissues. Excess host DNA can saturate the binding capacity of filters used in dot-blot pro cessing and therefore decrease the binding of par asite DNA.6 The recent resurgence of malaria7 and the anticipated vaccine studies89 have in tensified the search for methods which can be used for efficient large scale diagnosis of malaria. Specific and sensitive DNA probes could be used Accepted 31 December 1986. to define suitable vaccine study populations and to assess malaria control efforts. Several features make synthetic DNA probes derived from short, repetitive, species-specific sequences potentially useful in detecting eukary otic pathogens. Very pure synthetic DNA can be prepared inexpensively in large quantities. Syn thetic DNA lacks complex vector and nonre petitive parasite DNA sequences which may hybridize nonspecifically to the DNA of heter ologous species. The effective repetitive DNA concentration is higher than similar concentra tions of plasmid-borne or geonomic DNA, thus allowing shorter hybridization times.'° Because synthetic probes are single stranded, it is not nec essary to boil the probe before use, and synthetic DNA concentrations do not decrease during hy bridization due to reannealing with antiparallel probe sequences. Although signals from syn thetic probes are typically weaker than those from longer plasmid probes because of the absence of “tailing― or “networking―effects,― longer cx