DETECTION OF POLYMERASE CHAIN REACTION-AMPLIFIED HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 PROVIRAL DNA WITH A DIGOXIGENIN-LABELED RNA PROBE AND AN ENZYME-LINKED IMMUNOASSAY

An enzyme-linked immunoassay (EIA) combined with a solution hybridizat ion (SH) reaction was devised to detect human immunodeficiency virus t ype 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 labele d 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 f luorogenic substrate. The hybridization, immunological, and amplificat ion parameters of PCR-EIASH were optimized as follows: 12.5 pmol of ea ch primer was used in the PCR; the reannealing reaction of amplified p roducts with the RNA probe, which was used at 0.30 mug/ml, was complet ed in 30 min at 70-degrees-C in 2 x SSC (1 x SSC is 0.15 M NaCl plus 0 .015 M sodium citrate). Five copies of HIV-1 DNA diluted in a lysate o f 100,000 PBMCs from a seronegative control could be detected by PCR-E IASH with a signal of 41 +/- 3 fluorescent units above a background no ise of 13 +/- 2 fluorescent units. A total of 91 PBMC lysates from 91 seropositive patients sampled once and 20 PBMC lysates from 10 seropos itive patients sampled twice were tested in duplicate in the PCR-EIASH ; 107 samples were positive in duplicate tests, 1 sample was indetermi nate, and 3 samples were negative. Of the latter three samples, one be came positive by diluting the cell lysate, suggesting the presence of an inhibitor of Taq polymerase. The three samples negative for HIV-1 b y PCR-EIASH were also negative when amplified with SK145-SK39 and dete cted with P-32-labeled SK102.