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Amplification and detection of a single molecule of human immunodeficiency virus RNA

Abstract

Detection of plasma viremia in human immunodeficiency virus type 1 (HIV-1)-infected people is indispensable for the diagnosis of seronegative infection as well as for the evaluation of virus activities in vivo. The direct detection of HIV-1 RNA in circulation has been performed by means of reverse transcription followed by polymerase chain reaction (RT-PCR). As an attempt to establish a highly sensitive assay, we evaluated the effects of two-step amplification with nested primers and double priming of reverse transcription on the sensitivity of RT-PCR. The sensitivity of two-step amplification was 100 times higher than that of one-step amplification. The double priming of reverse transcription further increased the sensitivity of the following two-step amplification 100 times, which appeared to be enough to detect HIV-1 RNA from as little as a 2.2×10−4 TCID50 unit equivalent of culture supernatant of HIV-1-infected cells and a single molecule of HIV-1gag complementary RNA synthesized by in vitro transcription. By use of this most sensitive assay, we successfully detected HIV-1 RNA in serum or plasma 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 interferon-α or azidothymidine, 17 cases showed negative results, indicating that the virus activity was suppressed by the therapeutics. We also noted the inhibitory effect of heparin on RT-PCR.

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Correspondence to Dr. Shigeru Katamine.

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Nakamura, S., Katamine, S., Yamamoto, T. et al. Amplification and detection of a single molecule of human immunodeficiency virus RNA. Virus Genes 7, 325–338 (1993). https://doi.org/10.1007/BF01703389

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Key words

  • HIV
  • RNA
  • reverse transcription
  • polymerase chain reaction
  • heparin