Rapid in situ detection of PCR-amplified HIV-1 DNA.
The low copy number of human immunodeficiency virus 1 (HIV-1) DNA infected cells precludes routine detection by in situ hybridization. The inability to detect cells latently infected by HIV-1 makes difficult the study of factors that induce viral transcription, an essential factor in the development of the acquired immune deficiency syndrome (AIDS). A sensitive and rapid technique to detect HIV-1 DNA could be used as a diagnostic test for AIDS and to differentiate latent versus active viral infection. We describe a 3-h technique whereby HIV-1 DNA is amplified by hot start polymerase chain reaction (PCR) and detected directly in infected cells. The specificity of the assay was demonstrated by double labeling the positive cells with CD4. Using a CR10 HIV-1-infected cell line, the 90% of cells that were HIV-1 DNA positive could be distinguished from the 10% that were actively expressing HIV-1 RNA. The PCR in situ technique should allow for the direct localization of DNA sequences in cells that would otherwise be undetectable by conventional in situ analysis.
Department of Pathology, SUNY at Stony Brook 11794-8691.
G J
GJ
Nuovo
M
M
Margiotta
P
P
MacConnell
J
J
Becker
D000163:Acquired Immunodeficiency Syndrome / Q000175:diagnosis / Q000382:microbiology; D015496:CD4-Positive T-Lymphocytes / Q000382:microbiology; D002460:Cell Line; D004279:DNA, Viral / Q000097:blood* / Q000235:genetics*; D005069:Evaluation Studies as Topic; D015497:HIV-1 / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods* / Q000706:statistics & numerical data; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D012680:Sensitivity and Specificity; D013997:Time Factors
D004279:DNA, Viral