Enzyme immunoassay for detection of hybrids between PCR-amplified HIV-1 DNA and a RNA probe: PCR-EIA.
An enzyme immunoassay was developed to detect human immunodeficiency virus type 1 (HIV-1) DNA amplified by polymerase chain reaction (PCR-EIA). A set of primers (outer set) was used in PCR to amplify a segment of the HIV-1 gag gene from peripheral blood mononuclear cells. Hybrids between the amplified DNA and a RNA probe were measured in a microtiter plate immunoassay using a beta-D-galactosidase-conjugated monoclonal antibody to DNA-RNA hybrids and a fluorescent substrate. A second set of primers (nested set) located within the outer set was used in PCR with a known template to prepare the probe. One primer of the nested set included the T7 RNA polymerase promoter at its 5' end allowing transcription of a single-stranded RNA probe. Ten copies of HIV-1 DNA could be detected by PCR-EIA (42 fluorescent units with a background of 18 fluorescent units) compared with a detection limit of 1000 copies by ethidium bromide-stained agarose gel. HIV-1 DNA was detected by PCR-EIA in peripheral blood mononuclear cells from 32 of 33 seropositive patients (range 54-810 fluorescent units), and 0 of 25 seronegative patients (range 20-40 fluorescent units) (sensitivity 97%; specificity 100%). PCR-EIA offers a practical and nonisotopic method to objectively measure PCR-amplified HIV-1 DNA and has the potential for the measurement of other microbial pathogens in human body fluids.
Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
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Coutlée
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Yang
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Mayur
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Yolken
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D001483:Base Sequence; D004279:DNA, Viral / Q000097:blood*; D015658:HIV Infections / Q000097:blood / Q000175:diagnosis; D015497:HIV-1 / Q000302:isolation & purification*; D006801:Humans; D007124:Immunoenzyme Techniques; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D015347:RNA Probes
D004279:DNA, Viral; D015347:RNA Probes