Evaluation of infection with human immunodeficiency virus type 1 by using nonisotopic solution hybridization for detection of polymerase chain reaction-amplified proviral DNA

F. Coutlee, P. Saint-Antoine, C. Olivier, H. Voyer, A. Kessous-Elbaz, F. Barrada, P. Begin, L. Giroux, R. Viscidi

Research output: Contribution to journalArticlepeer-review

Abstract

A convenient assay combining solution hybridization and enzyme immunoassay for DNA-RNA hybrids (polymerase chain reaction-enzyme immunoassay [PCR-EIA]) was developed to detect human immunodeficiency virus type 1 (HIV-1) provirus amplified by the PCR and was compared with oligomer hybridization with 32P-labeled SK19. In PCR-EIA, a fragment of the HIV-1 gag gene from peripheral blood mononuclear cells was first amplified with primer pair SK38/SK39 or O1/O2. PCR-amplified material was reacted in solution with a biotinylated RNA probe. Biotinylated hybrids were measured in a microtiter-plate EIA with antibiotin antibody and a β-D-galactosidase-conjugated monoclonal antibody to DNA-RNA hybrids. Ten copies of HIV-1 DNA could be detected by PCR-EIA by using two different sets of primers. HIV-1 DNA was detected in 104 of 108 peripheral blood mononuclear cell samples by using SK38/39 and oligomer hybridization, in 104 of 108 samples by using SK38/SK39 and PCR-EIA, and in 104 of 108 samples by using O1/O2 and PCR-EIA. HIV-1 provirus was detected in 107 of 108 samples by using a combination of two sets of primers. One sample from a seropositive patient was negative in all three PCR assays, and six samples gave discordant results between primer pairs. Six of the latter samples scored negative in a PCR for β-globin but became positive when the sample was diluted before amplification. When applied to clinical samples, PCR-EIA generated results similar to those of an isotopic assay for detection of amplified DNA.

Original languageEnglish (US)
Pages (from-to)2461-2467
Number of pages7
JournalJournal of clinical microbiology
Volume29
Issue number11
DOIs
StatePublished - 1991
Externally publishedYes

ASJC Scopus subject areas

  • Microbiology (medical)

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