Important note: Information in this article was accurate in 1988. The state of the art may have changed since the publication date.
Direct detection of HIV-1 RNA from AIDS and ARC patient samples.
DNA. 1988 May;7(4):287-95. Unique Identifier : AIDSLINE MED/88283348 Murakawa GJ; Zaia JA; Spallone PA; Stephens DA; Kaplan BE; Wallace RB; Rossi JJ; Department of Microbiology, University of California, Los Angeles; 90024.
Abstract:
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.
Keywords: Acquired Immunodeficiency Syndrome/*DIAGNOSIS/MICROBIOLOGY AIDS-Related Complex/*DIAGNOSIS/MICROBIOLOGY Gene Amplification Genes, Viral Human HIV/*GENETICS/ISOLATION & PURIF Oligoribonucleotides/CHEMICAL SYNTHESIS Plasmids RNA, Viral/*ANALYSIS Support, Non-U.S. Gov't Support, U.S. Gov't, Non-P.H.S. Support, U.S. Gov't, P.H.S. Transcription, Genetic JOURNAL ARTICLE
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Important note: Information in this article was accurate in 1988. The state of the art may have changed since the publication date.
