Direct detection of HIV-1 RNA from AIDS and ARC patient samples.
Human immunodeficiency virus (HIV), formerly termed human T-lymphotropic virus (HTLVIII/LAV), is the etiological agent of acquired immune deficiency syndrome (AIDS). Direct detection of HIV-1 nucleic acid sequences in patient tissue or blood samples is possible in only a minor fraction of cases due to the low percentage of infected cells (Shaw et al., 1984). We report a modification of the polymerase chain reaction method (PCR) (Saiki et al., 1985), in which we amplify sequences from HIV-1 RNA templates, for the identification of HIV-1 in peripheral blood and tissue samples obtained from AIDS and AIDS-related complex (ARC) patients. This method of HIV-1 detection is at least six orders of magnitude more sensitive than standard nucleic acid detection methods and has direct clinical applications. In vitro tissue culturing of the virus is not required for HIV-1 detection. Using this technique, the sequence in the orfB region of HIV-1 has been amplified and detected from less than 1 microgram of total RNA prepared from a few milliliters of peripheral blood samples. This technique enables the rapid and unambiguous clinical detection of potential HIV-infected individuals and can be used to assay the efficacy of anti-HIV-1 drugs. To enhance the efficiency of this technique, we have appended the prokaryotic T7 RNA polymerase promoter sequence to one of the priming oligonucleotides. After several cycles of PCR with the promoter-containing oligo, a small aliquot of the reaction can be utilized to direct specific and efficient T7 RNA polymerase-mediated transcription of the amplified sequences, thus enhancing the sensitivity and simplifying the labor of the experiment.
Murakawa
G J
GJ
Department of Microbiology, University of California, Los Angeles 90024.
Zaia
J A
JA
Spallone
P A
PA
Stephens
D A
DA
Kaplan
B E
BE
Wallace
R B
RB
Rossi
J J
JJ
D000386:AIDS-Related Complex / Q000175:diagnosis* / Q000382:microbiology; D000163:Acquired Immunodeficiency Syndrome / Q000175:diagnosis* / Q000382:microbiology; D005784:Gene Amplification; D005814:Genes, Viral; D006678:HIV / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D009843:Oligoribonucleotides / Q000138:chemical synthesis; D010957:Plasmids; D012367:RNA, Viral / Q000032:analysis*; D014158:Transcription, Genetic
D009843:Oligoribonucleotides; D012367:RNA, Viral