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  • Title: DNA probe hybridisation in microwells using a new bioluminescent system for the detection of PCR-amplified HIV-1 proviral DNA.
    Author: Zammatteo N, Moris P, Alexandre I, Vaira D, Piette J, Remacle J.
    Journal: J Virol Methods; 1995 Oct; 55(2):185-97. PubMed ID: 8537457.
    Abstract:
    A new bioluminescent detection system combined with a sandwich DNA hybridisation reaction in microwells has been developed for the detection of human immunodeficiency virus type 1 (HIV-1) provirus DNA amplified by the polymerase chain reaction (PCR). First, a fragment of the HIV-1 gag gene was amplified. The amplified DNA fragments were denatured and hybridised to a capture probe immobilised in microwells and to a biotinylated detection probe. A streptavidin-pyruvate kinase conjugate could then react on the biotinylated probe and the kinase activity detected by means of the luciferin-luciferase system, with production of a bioluminescent signal. This sandwich assay followed by a bioluminescent reaction detected as little as 7 amol of target DNA. The bioluminescent assay detected 5 HIV copies generated after one round of PCR, even if no band was seen on an agarose gel. The assay was applied to the detection of HIV-proviral DNA in peripheral blood mononuclear cells after one round of PCR and allowed to clearly identify a positive sample as compared to nested PCR.
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