DNA probe hybridisation in microwells using a new bioluminescent system for the detection of PCR-amplified HIV-1 proviral DNA.
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.
Zammatteo
N
N
Laboratoire de Biochimie Cellulaire, Facultés Notre-Dame de la Paix, Namur, Belgium.
Moris
P
P
Alexandre
I
I
Vaira
D
D
Piette
J
J
Remacle
J
J
D001483:Base Sequence; D017931:DNA Primers; D015342:DNA Probes; D004279:DNA, Viral / Q000032:analysis*; D015683:Gene Products, gag / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000821:virology; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011533:Proviruses / Q000235:genetics / Q000302:isolation & purification; D012680:Sensitivity and Specificity
D017931:DNA Primers; D015342:DNA Probes; D004279:DNA, Viral; D015683:Gene Products, gag