DETECTION OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 PROVIRAL DNA BY PCR USING AN ELECTROCHEMILUMINESCENCE-TAGGED PROBE

We have developed a rapid, pseudohomogeneous assay for the detection o f PCR amplicons, based on the use of electrochemiluminescence generate d from a Tris-bipyridine ruthenium) label. PCR amplification of highly conserved human immunodeficiency virus type 1 (HIV-1) gag gene sequen ces 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, dena tured by heating at 100 degrees C for 5 min, and hybridized at 55 degr ees C for an additional 15 min. Hybridization to the biotinylated stra nd of the amplified DNA was determined by the addition of streptavidin -conjugated magnetic particles and analyzed by using an Origen-1 elect rochemiluminescence analyzer. Our results demonstrated a sensitivity o f 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 l ysed, proteinase K-treated 8E5 cells as the starting material. In an e valuation of actual clinical specimens (peripheral blood monocytes fro m both healthy and HIV-1-infected children), the electrochemiluminesce nt detection assay correlated 100% with both our standard method (solu tion hybridization with a radiolabeled probe followed by polyacrylamid e gel electrophoresis [PAGE] and autoradiography) and a commercial met hod (Roche Amplicor). The electrochemiluminescent method was substanti ally easier to perform than either the PAGE or microtiter plate assays and was considerable faster to perform than either of these alternati ve formats.