Detection of HIV-1 by digoxigenin-labelled PCR and microtitre plate solution hybridisation assay and prevention of PCR carry-over by uracil-N-glycosylase.
An extremely sensitive and convenient microtiter plate solution hybridisation assay for the detection of HIV-1 PCR products was developed. The PCR product is labelled by direct incorporation of digoxigenin-dUTP and after denaturation is captured by a microtitre plate coated with a streptavidin-linked biotinylated probe. The PCR/probe hybrids are reacted with an alkaline phosphate conjugated anti-digoxigenin antibody and detected using an alkaline phosphatase enzyme amplification system. The use of uracil-N-glycosylase and dUTP instead of dTTP in the PCR is used to effectively control carry-over from previous PCR products. The assay can detect single HIV-1 DNA molecules in a background DNA of 0.75 microgram.
King
J A
JA
Regional Virus Laboratory, East Birmingham Hospital, UK.
Ball
J K
JK
D001483:Base Sequence; D045647:DNA Glycosylases; D017931:DNA Primers; D015342:DNA Probes; D016373:DNA, Antisense; D004279:DNA, Viral / Q000032:analysis*; D003856:Deoxyuracil Nucleotides; D004076:Digoxigenin; D015752:Genes, env; D015750:Genes, gag; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D008832:Microchemistry / Q000295:instrumentation; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases; D009693:Nucleic Acid Hybridization / Q000295:instrumentation / Q000379:methods*; D016133:Polymerase Chain Reaction; D011533:Proviruses / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D051981:Uracil-DNA Glycosidase
D017931:DNA Primers; D015342:DNA Probes; D016373:DNA, Antisense; D004279:DNA, Viral; D003856:Deoxyuracil Nucleotides; C027078:deoxyuridine triphosphate; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase; D004076:Digoxigenin