[Show abstract][Hide abstract] ABSTRACT: The polymerase chain reaction (PCR) fails to detect HIV-1 sequences in 5% of infected individuals. To screen for false-negative PCR tests, we developed a nonisotopic PCR assay in which sequences from the beta-globin gene and from the HIV-1 vpu-env region were coamplified with biotinylated and fluorescein-labeled primers, respectively. Coamplified products were reacted with specific internal digoxigenin-labeled RNA probes. Hybrids were detected in a microtiter plate coated with streptavidin or anti-fluorescein antibody, with enzyme-labeled anti-digoxigenin antibody. After the optimization of the coamplification conditions, the assay could detect 5 proviral DNA copies in a lysate from 100,000 peripheral blood mononuclear cells. Fifty-seven samples from 55 HIV-1-seropositive patients and 25 samples from 25 seronegative individuals were evaluated. Fifty-two samples from HIV-infected individuals were positive for HIV-1 vpu-env sequences. Three of the 5 PBMC lysates falsely negative for HIV-1 sequences had reactivities for beta-globin (3-23 fu) below that of 100,000 cells (304 fu). Nonisotopic coamplification allowed for the evaluation of the quality of specimens for PCR concurrently with the detection of the presence of viral template sequences.
No preview · Article · Apr 1995 · AIDS Research and Human Retroviruses
[Show abstract][Hide abstract] ABSTRACT: An enzyme-linked immunoassay (EIA) combined with a solution hybridization (SH) reaction was devised to detect human immunodeficiency virus type 1 (HIV-1) provirus amplified by the polymerase chain reaction (PCR). In this nonisotopic PCR assay, designated PCR-EIASH, a fragment of the HIV-1 gag gene from peripheral blood mononuclear cells (PBMCs) was first amplified with biotinylated primers. The biotinylated amplified DNA segment was reacted in solution with an internal RNA probe labeled with digoxigenin-11-UTP. Hybrids were captured in a microtiter plate coated with streptavidin. Specific bound hybrids were quantitated by the addition of an enzyme-labeled antibody against digoxigenin and a fluorogenic substrate. The hybridization, immunological, and amplification parameters of PCR-EIASH were optimized as follows: 12.5 pmol of each primer was used in the PCR; the reannealing reaction of amplified products with the RNA probe, which was used at 0.30 microgram/ml, was completed in 30 min at 70 degrees C in 2x SSC (1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate). Five copies of HIV-1 DNA diluted in a lysate of 100,000 PBMCs from a seronegative control could be detected by PCR-EIASH with a signal of 41 +/- 3 fluorescent units above a background noise of 13 +/- 2 fluorescent units. A total of 91 PBMC lysates from 91 seropositive patients sampled once and 20 PBMC lysates from 10 seropositive patients sampled twice were tested in duplicate in the PCR-EIASH; 107 samples were positive in duplicate tests, 1 sample was indeterminate, and 3 samples were negative. Of the latter three samples, one became positive by diluting the cell lysate, suggesting the presence of an inhibitor of Taq polymerase. The three samples negative for HIV-1 by PCR-EIASH were also negative when amplified with SK145-SK39 and detected with 32P-labeled SK102.
Preview · Article · Jun 1993 · Journal of Clinical Microbiology