Polymerase chain reaction and a liquid-phase, nonisotopic hybridization for species-specific and sensitive detection of malaria infection

Abstract

In the present study, we describe a polymerase chain reaction (PCR)-based enzyme-linked immunosorbent assay for the detection of malaria infection. The target region of the 18S ribosomal DNA is amplified by a PCR using an 18S rRNA, genus-specific, biotinylated (5') and an unlabeled primer (3') pair. The detection probes are digoxigenin-labeled DNA oligonucleotides derived from species-specific rRNA sequences. The amplified fragments are allowed to hybridize with the species-specific, digoxigenin-labeled oligonucleotide probes. The oligo/DNA complex is allowed 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, specific, simple, and reliable, with wide applicability in epidemiologic studies, accurate detection of mixed infections, detection of low-level parasitemia, and evaluation of chemotherapy and vaccine efficacy.