Document 0838
 DOCN  M9480838
 TI    Detection of HIV-1 proviral DNA by polymerase chain reaction using an
       electrochemiluminescent-tagged probe.
 DT    9410
 AU    Schutzbank TE; Smith J; Greeley C; Children's Hospital National Medical
       Center, Washington, D.C.
 SO    Abstr Gen Meet Am Soc Microbiol. 1994;94:555 (abstract no. C-367).
       Unique Identifier : AIDSLINE ASM94/94313107
 AB    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.
 DE    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  MEETING ABSTRACT

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).