AMPLIFICATION AND DETECTION OF A SINGLE-MOLECULE OF HUMAN-IMMUNODEFICIENCY-VIRUS RNA

Detection of plasma viremia in human immunodeficiency virus type 1 (HI V-1)-infected people is indispensable for the diagnosis of seronegativ e infection as well as for the evaluation of virus activities in vivo. The direct detection of HIV-1 RNA in circulation has been performed b y means of reverse transcription followed by polymerase chain reaction (RT-PCR). As an attempt to establish a highly sensitive assay, we eva luated the effects of two-step amplification with nested primers and d ouble priming of reverse transcription on the sensitivity of RT-PCR. T he sensitivity of two-step amplification was 100 times higher than tha t of one-step amplification. The double priming of reverse transcripti on further increased the sensitivity of the following two-step amplifi cation 100 times, which appeared to be enough to detect HIV-1 RNA from as little as a 2.2 x 10(-4) TCID50 unit equivalent of culture superna tant of HIV-1-infected cells and a single molecule of HIV-1 gag comple mentary RNA synthesized by in vitro transcription. By use of this most sensitive assay, we successfully detected HIV-1 RNA in serum or plasm a from all 22 patients with acquired immune deficiency syndrome (AIDS) or AIDS-related complex (ARC) and 13 out of 14 untreated asymptomatic carriers. Of 43 asymptomatic carriers under the treatment with interf eron-a or azidothymidine, 17 cases showed negative results, indicating that the virus activity was suppressed by the therapeutics. We also n oted the inhibitory effect of heparin on RT-PCR.