Sensitive and quantitative detection of PCR-amplified HIV-1 DNA products by an enzyme linked immunoassay following solution hybridization with two differently labelled oligonucleotide probes.
We have developed and evaluated an ELISA-based detection method for PCR-amplified HIV-1 DNA. The assay uses two oligonucleotide probes which are end-labelled at the 5'-end with biotin or digoxigenin, respectively. Upon solution hybridization of these probes which react with the same strand of amplified DNA product, the formed hybrids are bound to avidin-coated wells of a microtitre plate and detected by horseradish peroxidase-labelled antibodies directed against digoxigen and 3, 3', 5, 5'-tetramethylbenzidine as substrate. Factors critical for a high signal-to-noise ratio were the use of serum as blocking agent, the amount of biotin-labelled and digoxigenin-labelled oligonucleotide probes present in a reaction and the inactivation of Taq DNA polymerase. The method has a detection limit of 1-3 pg of amplified DNA and is quantitative in a range extending from 1 pg to at least 200 pg. In the background of 1 microgram of total DNA, one single-stranded copy of HIV-1 DNA can be detected after 35 cycles of amplification. A comparison of the ELISA-based detection method with primer extension analysis, a method previously shown to reach a similar detection limit, demonstrated complete agreement of the results of 118 amplified DNAs. The method proved simple, requires only about 3 h, and could easily be adapted to the detection of other amplified target DNAs.
Böni
J
J
Swiss National Center for Retroviruses, Institute for Medical Virology, University of Zurich.
Schüpbach
J
J
D001483:Base Sequence; D001710:Biotin; D002460:Cell Line; D017931:DNA Primers; D004279:DNA, Viral / Q000302:isolation & purification; D004076:Digoxigenin; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D015750:Genes, gag; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D017931:DNA Primers; D004279:DNA, Viral; D015345:Oligonucleotide Probes; D001710:Biotin; D004076:Digoxigenin