A rapid and sensitive PCR assay for detection of HIV-1 specific DNA and RNA in serum and culture supernatants was developed. Normal serum spiked with various amounts of HIV-1 containing plasmid BH10 or supernatant from infected cells was analyzed. Samples were treated with 0.1% NP40 and heated at 70 C for 5-10 minutes to remove cellular debris. An aliquot was amplified by PCR using primer pairs from the gag, env and nef region of the viral genome in a co-amplification. For detection of RNA, samples were treated with a guanidine thiocyanate based buffer followed by phenol/CHC13 extraction. RNA was precipitated with isopropanol and analyzed by PCR. RNA was amplified by conversion to cDNA by reverse transcriptase followed by amplification with Taopolymerase. PCR products were analyzed by slot- blots or liquid hybridization with radiolabeled oligonucleotide probes followed by PAGE and autoradiography. By this method as little as 10- 20pies of plasmid DNA could be detected. Viral DNA or RNA present in as little as 10 ul of serum from an AIDS patient or less than 0.1 ul of culture supernatant from infected H9 cells. No reactivity was observed in unspiked control samples of serum or uninfected H9 cell cell culture fluid. This assay may be useful in assessing plasma viremia and in vitro studies.
