Detection of HIV-1 proviral DNA by polymerase chain reaction using an electrochemiluminescent-tagged probe.

Important note: Information in this article was accurate in 1994. The state of the art may have changed since the publication date.

Detection of HIV-1 proviral DNA by polymerase chain reaction using an electrochemiluminescent-tagged probe.

Abstr Gen Meet Am Soc Microbiol. 1994;94:555 (abstract no. C-367). Unique Identifier : AIDSLINE ASM94/94313107
Schutzbank TE; Smith J; Greeley C; Children's Hospital National Medical Center, Washington, D.C.

Abstract: We have developed a rapid, pseudo-homogeneous assay for the detection of PCR amplicons, based on the use of electroluminescence generated from a Tris-bipyridine ruthinium (II) (Ru(bpy)3(3+)) label. PCR amplification of highly conserved HIV-1 gag gene sequences were performed using SK38/SK39 primers, where the latter was 5'-biotinylated. Post-PCR reactions were mixed with 10(12) copies of the SK19 probe-Ru(bpy)2(3+) NHS ester conjugate, denatured by heating at 100 degrees C for 5 minutes, and hybridized at 55 degrees C for an additional 15 minutes. Hybridization to the biotinylated strand of the amplified DNA was determined by the addition of streptavidin-conjugated magnetic particles, and analysis on an Origen-1 electroluminescence analyzer. Our results demonstrated a sensitivity of less than 5 copies of HIV-1 (pre-PCR), using either purified plasmid DNA containing one complete copy of the HIV-1 cDNA genome, or lysed, proteinase K treated 8E5 cells as the starting material. In an evaluation with actual clinical specimens, (peripheral blood monocytes from both normal and HIV-1-infected children), the electroluminescent detection assay correlated 100% with our standard method (solution hybridization with a radiolabeled probe followed by polyacrylamide gel electrophoresis [PAGE] and autoradiography). The electroluminescent method was substantially easier to perform than either PAGE or microtiter plate assays, and was considerable faster than either of these alternative formats.

Keywords: Acquired Immunodeficiency Syndrome/BLOOD/*DIAGNOSIS/IMMUNOLOGY Base Sequence Chemiluminescence Child Conserved Sequence DNA Primers DNA, Viral/*BLOOD Electrochemistry/*METHODS Electrophoresis, Polyacrylamide Gel *Genes, gag Human HIV-1/GENETICS/*ISOLATION & PURIF Monocytes/MICROBIOLOGY Oligonucleotide Probes Polymerase Chain Reaction/*METHODS Proviruses/GENETICS/*ISOLATION & PURIF Reference Values ABSTRACT
941030
M94A0847

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