Construction of appropiate DNA probes of Plasmodium falciparum for detection of infected mosquitoes

Abstract

DNA from Plasmodium falciparum, Thai isolate K1, was purified and fragmented using restriction endonuclease EcoRI*. These DNA fragments were inserted into pUN21 at the EcoRI site and cloned in E. coli strain JM 107. A library containing 20,000 tetracycline-resistant transformants were obtained. Using colony hybridization technique, 53 recombinant DNA clones were selected. From Southern hybridization of 53 recombinant plasmids, pUNK1-32, -34, -43, -45, -51 were selected. The two sensitive recombinant DNA probes. pUNK1-34 and pUNK1-45, were able to detect a parasitemia of at least 0.005% in 20 μl of infected blood. pUNK1-45 did not cross-hybridize with DNA from other plasmodial species, eg. P. chabaudi, P. knowlesi and P. cynomolgi nor with man and mosquito. The plasmid pUNK1-34 did not cross-hybridize with other DNA except from P. chabaudi, Using pUNK1-45 as probe, oocysts and sporozoites could be detected in only one or less infected mosquito. The probe was able to detect at least 5000-1000 sporozoites. Studies of restriction map and organization of the DNA inserts of pUNK1-34 and pUNK1-45 in genome of P.falciparum showed that the two fragments were not homologous. The estimated copy number of pUNK 1-34 and pUNK1-45 in the genome of P. falciparum was about 25 and 120.