Amplification and detection of a single molecule of human immunodeficiency virus RNA.
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 x 10(-4) TCID50 unit equivalent of culture supernatant of HIV-1-infected cells and a single molecule of HIV-1 gag 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-alpha 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.
Nakamura
S
S
Department of Bacteriology, Nagasaki University School of Medicine, Japan.
Katamine
S
S
Yamamoto
T
T
Foung
S K
SK
Kurata
T
T
Hirabayashi
Y
Y
Shimada
K
K
Hino
S
S
Miyamoto
T
T
D001483:Base Sequence; D002478:Cells, Cultured; D004279:DNA, Viral; D015683:Gene Products, gag / Q000235:genetics; D015658:HIV Infections / Q000175:diagnosis / Q000382:microbiology*; D006679:HIV Seropositivity / Q000175:diagnosis / Q000382:microbiology; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis* / Q000097:blood; D012680:Sensitivity and Specificity; D014766:Viremia / Q000175:diagnosis / Q000382:microbiology*
D004279:DNA, Viral; D015683:Gene Products, gag; D012367:RNA, Viral