Incorporation of biotinylated nucleotides for the quantification of PCR-amplified HIV-1 DNA by chemiluminescence.
Chemiluminescent detection of polymerase chain reaction (PCR-)amplified DNA was used as a quantitative method for detecting HIV-1. For this purpose, biotinylated dUMP was directly incorporated into the amplified DNA during the PCR reaction. Biotinylation was visualized in an enzymatic reaction using avidine-conjugated alkaline phosphatase and its chemiluminescent 1,2-dioxetane substrate AMPPD, which decomposes upon dephosphorylation and emits light. Light emission was either detected with X-ray films or quantified with a single-photon counting camera connected to a computer imaging system. The specificity of the method was shown by hybridization with a biotinylated or radiolabelled HIV-1-specific oligonucleotide probe. Besides being quantitative, this method represented a non-hazardous, rapid and sensitive technique for the detection of HIV-1 DNA.
Bettens
F
F
Institut für klinische Immunologie, Inselspital Bern.
Pichler
W J
WJ
de Weck
A L
AL
D001710:Biotin / Q000737:chemistry*; D004279:DNA, Viral / Q000032:analysis*; D003856:Deoxyuracil Nucleotides / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D015750:Genes, gag / Q000235:genetics; D015658:HIV Infections / Q000175:diagnosis; D015497:HIV-1 / Q000235:genetics*; D008163:Luminescent Measurements; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction / Q000379:methods*
D004279:DNA, Viral; D003856:Deoxyuracil Nucleotides; D015345:Oligonucleotide Probes; D001710:Biotin; C007267:2'-deoxyuridylic acid