Confirmation of HIV infection using gene amplification.
Direct recognition of viral gene sequences can be used to detect human immunodeficiency virus (HIV-1) in clinical specimens. A modification of the polymerase chain reaction (PCR) for amplification of gene sequences was used for detection of HIV-1-specific RNA prepared from peripheral blood mononuclear cells (PBMC). The RNA served as a template for reverse transcriptase using primers derived from both the 3'ORF and the LTR regions of HIV-1, as well as from the control cellular sequences encoding beta-actin and T cell receptor. The resultant DNA was amplified with DNA polymerase. A transcriptional step using the bacteriophage T7 promoter recognition sequences, incorporated into the primers, was used to enhance the efficiency of the amplification process. This assay detects as few as 100 RNA copies of cloned HIV-1 genome. Starting with 1 microgram RNA isolated from PBMC, we were able to detect HIV-1 sequences in patients with symptomatic and asymptomatic HIV-1 infection. The inclusion of T cell-specific primers permitted simultaneous evaluation of an immunologic parameter. The PCR can be applied to RNA samples for detection of viral and cellular sequences and is a rapid and efficient means for detection of HIV-1 sequences as well as potentially informative cellular sequences.
Zaia
J A
JA
City of Hope National Medical Center, Durate, CA 91010.
Rossi
J J
JJ
D015153:Blotting, Western; D015497:HIV-1 / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000302:isolation & purification
D012367:RNA, Viral