title: Detection of human immunodeficiency virus type 1 proviral DNA by PCR using an electrochemiluminescence-tagged probe.
abstract: 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.
authors.affiliation: Children's National Medical Center, Washington, D.C., USA.
authors.forename: T E
authors.initials: TE
authors.lastname: Schutzbank
authors.forename: J
authors.initials: J
authors.lastname: Smith
mesh_terms: D002648:Child; D002675:Child, Preschool; D015342:DNA Probes; D004279:DNA, Viral / Q000235:genetics* / Q000302:isolation & purification*; D004563:Electrochemistry; D004591:Electrophoresis, Polyacrylamide Gel; D005069:Evaluation Studies as Topic; D015658:HIV Infections / Q000175:diagnosis / Q000821:virology; D015497:HIV-1 / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D008163:Luminescent Measurements; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000706:statistics & numerical data; D011533:Proviruses / Q000235:genetics* / Q000302:isolation & purification*; D012680:Sensitivity and Specificity
chemical_list: D015342:DNA Probes; D004279:DNA, Viral