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.