Probes for quantitating subpicogram amounts of HIV-1 RNA by molecular hybridization.
A set of probes was designed for the quantitation of HIV-1 RNA in infected cells by a molecular hybridization procedure called reversible target capture. Reversible target capture is analogous to sandwich hybridization, except that the link between hybrid complexes and the affinity support was reversible, allowing for repeated capture of hybrids on, and release from, fresh affinity support. Repeated cycles of capture resulted in a high degree of purification of hybrids from unreacted probe, thereby greatly reducing assay noise and increasing assay sensitivity. Probes against the HIV-1 pol gene were chosen because their target sequences were highly conserved among HIV-1 isolates, while being divergent enough to provide discrimination from other human T cell tropic viruses. Subpicogram quantities of HIV-1 pol gene RNA were measured with signal:noise ratios of over 10. Hybridization signal increased with increasing target RNA with a proportionality constant of 1.
Gillespie
D
D
Hahnemann University, Department of Neoplastic Diseases, Philadelphia, PA 19102.
Thompson
J
J
Solomon
R
R
D002478:Cells, Cultured; D005814:Genes, Viral; D006678:HIV / Q000032:analysis*; D006801:Humans; D009693:Nucleic Acid Hybridization; D015347:RNA Probes; D012367:RNA, Viral / Q000032:analysis*; D012194:RNA-Directed DNA Polymerase / Q000235:genetics; D013601:T-Lymphocytes / Q000382:microbiology
D015347:RNA Probes; D012367:RNA, Viral; D012194:RNA-Directed DNA Polymerase