Enzyme Immunoassay for Detection of Hybrids Between PCR-Amplified HIV-1 DNA and a RNA Probe: PCR-EIA

François Coutlée, Bingzhi Yang, Linda Bobo, Kumudini Mayur, Robert Yolken, Raphael Viscidi

Research output: Contribution to journalArticle

Abstract

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 β-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.

Original languageEnglish (US)
Pages (from-to)775-784
Number of pages10
JournalAIDS research and human retroviruses
Volume6
Issue number6
DOIs
StatePublished - Jun 1990

ASJC Scopus subject areas

  • Immunology
  • Virology
  • Infectious Diseases

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