PLASMODIUM - GENUS-CONSERVED PRIMERS FOR SPECIES IDENTIFICATION AND QUANTITATION

Stable RNAs have regions of primary sequence that are nearly identical in every member of the Plasmodium genus and not found in the host or in other common pathogens. Several ''genus-conserved'' sequences, whic h flank hypervariable regions, were identified within the small subuni t ribosomal RNA of Plasmodium species. Primers based on these conserve d sequences permit amplification of species- or possibly even strain-s pecific sequences from samples of unknown composition. As an example o f this approach, sequences from the four human malaria species were su ccessfully recovered from Giemsa-stained blood smears, including two d ifferent sequences for Plasmodium ovale (of 91.5% similarity). This ty pe of information is useful for epidemiological and phylogenetic analy sis of any malaria species. We show that amplification of rRNA-derived sequences behaves in a competitive fashion during the cycles of polym erase amplification and therefore target sequences from Plasmodium spe cies are amplified in proportion to their abundance in the sample. The re are several implications of this finding. (1) The proportion of dif ferent products resulting from amplification from samples with mixed i nfections is closely related to the proportion of infecting species, ( 2) Direct quantitation of parasite nucleic acids within a sample can b e derived when known amounts of competitor RNA are added to the RT/PCR reaction. (3) Amplification of rRNA sequences, using genus-specific p rimers, allows one to monitor the development of the parasite in the m osquito. (C) 1995 Academic Press, Inc.