Polymerase Chain Reaction and a Liquid-Phase, Nonisotopic Hybridization for Species-Specific and Sensitive Detection of Malaria Infection

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  • Immunology Branch, Division of Parasitic Diseases, National Center for Infectious Diseases, Biotechnology Core Facility, Scientific Resources Program, and Division of Viral and Rickettsial Diseases, Centers for Disease Control and Prevention, Institute of Biomedical Sciences, University of Sao Paulo, Atlanta, Georgia

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.