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)-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.
Oliveira
D A
DA
Immunology Branch, National Center for Infectious Diseases, Atlanta, Georgia.
Holloway
B P
BP
Durigon
E L
EL
Collins
W E
WE
Lal
A A
AA
D000818:Animals; D001029:Aotus trivirgatus; D001483:Base Sequence; D003124:Colorimetry; D017124:Conserved Sequence; D017931:DNA Primers / Q000737:chemistry; D016054:DNA, Protozoan / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D004275:DNA, Ribosomal / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008288:Malaria / Q000175:diagnosis*; D016778:Malaria, Falciparum / Q000175:diagnosis*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000737:chemistry; D002679:Pan troglodytes; D010961:Plasmodium / Q000235:genetics / Q000302:isolation & purification*; D010963:Plasmodium falciparum / Q000235:genetics / Q000302:isolation & purification; D010965:Plasmodium malariae / Q000235:genetics / Q000302:isolation & purification; D010966:Plasmodium vivax / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D012337:RNA, Ribosomal, 18S / Q000737:chemistry / Q000235:genetics*; D012680:Sensitivity and Specificity; D013045:Species Specificity
D017931:DNA Primers; D016054:DNA, Protozoan; D004275:DNA, Ribosomal; D015345:Oligonucleotide Probes; D012337:RNA, Ribosomal, 18S