POLYMERASE CHAIN-REACTION AND A LIQUID-PHASE, NONISOTOPIC HYBRIDIZATION FOR SPECIES-SPECIFIC AND SENSITIVE DETECTION OF MALARIA INFECTION

In the present study, we describe a polymerase chain reaction (PCR)-ba sed enzyme-linked immunosorbent assay for the detection of malaria inf ection. The target region of the 18S ribosomal DNA is amplified by a P CR using an 18S rRNA, genus-specific, biotinylated (5') and an unlabel ed primer (3') pair. The detection probes are digoxigenin-labeled DNA oligonucleotides derived from species-specific rRNA sequences. The amp lified fragments are allowed to hybridize with the species-specific, d igoxigenin-labeled oligonucleotide probes. The oligo/DNA complex is al lowed to bind onto streptavidin-coated microtiter plates, followed by incubation with a peroxidase-streptavidin conjugate and a colorimetric -peroxidase substrate. The resulting test demonstrated specificity for the four human Plasmodium species, and was able to detect a level of parasitemia of at least 0.0001% in a laboratory-induced P. falciparum infection in monkeys, This liquid hybridization assay is sensitive, sp ecific, simple, and reliable, with wide applicability in epidemiologic studies, accurate detection of mixed infections, detection of low-lev el parasitemia, and evaluation of chemotherapy and vaccine efficacy.