       Document 0446
 DOCN  M9610446
 TI    Detection of human immunodeficiency virus type 1 proviral DNA by PCR
       using an electrochemiluminescence-tagged probe.
 DT    9601
 AU    Schutzbank TE; Smith J; Children's National Medical Center, Washington,
       D.C., USA.
 SO    J Clin Microbiol. 1995 Aug;33(8):2036-41. Unique Identifier : AIDSLINE
       MED/96057582
 AB    We have developed a rapid, pseudohomogeneous assay for the detection of
       PCR amplicons, based on the use of electrochemiluminescence generated
       from a Tris-bipyridine ruthenium(II) label. PCR amplification of highly
       conserved human immunodeficiency virus type 1 (HIV-1) gag gene sequences
       was performed with SK38 and SK39 primers, the latter of which was 5'
       biotinylated. Post-PCR reaction mixtures were combined with 10(12)
       copies of the SK19 probe-Tris-bipyridine ruthenium(II) conjugate,
       denatured by heating at 100 degrees C for 5 min, and hybridized at 55
       degrees C for an additional 15 min. Hybridization to the biotinylated
       strand of the amplified DNA was determined by the addition of
       streptavidin-conjugated magnetic particles and analyzed by using an
       Origen-1 electrochemiluminescence analyzer. Our results demonstrated a
       sensitivity of fewer than five copies of HIV-1 (pre-PCR), by 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 of actual clinical specimens (peripheral
       blood monocytes from both healthy and HIV-1-infected children), the
       electrochemiluminescent detection assay correlated 100% with both our
       standard method (solution hybridization with a radiolabeled probe
       followed by polyacrylamide gel electrophoresis [PAGE] and
       autoradiography) and a commercial method (Roche Amplicor). The
       electrochemiluminescent method was substantially easier to perform than
       either the PAGE or microtiter plate assays and was considerable faster
       to perform than either of these alternative formats.
 DE    Chemiluminescence  Child  Child, Preschool  Comparative Study  DNA
       Probes  DNA, Viral/*GENETICS/*ISOLATION & PURIF  Electrochemistry
       Electrophoresis, Polyacrylamide Gel  Evaluation Studies  Human  HIV
       Infections/DIAGNOSIS/VIROLOGY  HIV-1/*GENETICS/*ISOLATION & PURIF
       Infant  Infant, Newborn  Polymerase Chain Reaction/*METHODS/STATISTICS &
       NUMER DATA  Proviruses/*GENETICS/*ISOLATION & PURIF  Sensitivity and
       Specificity  Support, U.S. Gov't, P.H.S.  JOURNAL ARTICLE

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

