Evaluation of infection with human immunodeficiency virus type 1 by using nonisotopic solution hybridization for detection of polymerase chain reaction-amplified proviral DNA.
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 beta-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 beta-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.
Coutlée
F
F
Department of Microbiology and Infectious Diseases, Hopital Notre-Dame, Montreal, Quebec, Canada.
Saint-Antoine
P
P
Olivier
C
C
Voyer
H
H
Kessous-Elbaz
A
A
Berrada
F
F
Bégin
P
P
Giroux
L
L
Viscidi
R
R
D001483:Base Sequence; D001710:Biotin; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D005069:Evaluation Studies as Topic; D015658:HIV Infections / Q000175:diagnosis* / Q000235:genetics; D015497:HIV-1 / Q000235:genetics; D006801:Humans; D007124:Immunoenzyme Techniques; D008297:Male; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction / Q000379:methods*; D011533:Proviruses / Q000235:genetics / Q000302:isolation & purification; D015347:RNA Probes
D004279:DNA, Viral; D015347:RNA Probes; D001710:Biotin