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 C for 5 min, and hybridized at 55 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.

ECL—Electrochemical luminescence

“…Typically, these involve the hybridization and labeling of specific nucleic acid sequences to test for disease states, predisposition for a disease and infectious diseases. Assays for a variety of analytes have been reported in the areas of infectious diseases, [353][354][355][356][357][358][359][360][361][362][363][364][365][366][367][368][369][370] tumor markers, [371][372][373][374] metabolism, 375 venous thromboembolism, 376 and cystic fibrosis 377 (Table S6, ESIw). Several studies have coupled ECL with polymerase chain reaction (PCR) amplification to lower detection limits and increase sensitivity.…”

Infection of human primary renal epithelial cells with HIV-1 from children with HIV-associated nephropathy

“…Electrochemiluminescent detection of the HIV-1 specific amplification products were measured as described by Schutzbank and Smith [39]. Briefly, cells were removed and suspended in 250 l of lysis buffer (50 mM KCl, 10 mM Tris-HCl, pH 8.3, 2.5 mM MgCl, 0.45% Nonidet P-40, 0.45% Tween 20, and 10 g/ml proteinase K).…”

Rapid and cost-effective detection of sequence-specific DNA by monitoring the electrochemical response of 2′-deoxyguanosine 5′-triphosphate in a PCR sample

“…In the performance of sequence-specific DNA detection, polymerase chain reaction (PCR) is studied and employed most widely owing to its high sensitivity of fewer than five copies (even one copy) and many other advantages. 1,2 The technology of detecting PCR products is advancing the development of the whole kit to be more and more satisfactory. To date, the realtime PCR that employs fluorescent probes has established itself as the state-of-the-art technology.…”