Enhanced chemiluminescence-based hybridization analysis for PCR-mediated HIV-1 DNA detection offers an alternative to 32P-labelled probes.
The efficiency of enhanced chemiluminescence based on a novel generation substrate for alkaline phosphatase, adamantyl-1,2-dioxetane phosphate, was compared with that of 32P-labelled probe for visualization of human immunodeficiency virus type 1 (HTV-1)-specific DNA-DNA hybrids. The probe used for nonisotopic detection was digoxigenin labelled and targeted by anti-digoxigenin antibody Fab-fragments conjugated to alkaline phosphatase. The dot-blot hybridization analysis performed on a dilution series of HIV-1 proviral DNA demonstrated a lower sensitivity limit of 0.5 pg with the nonisotopic method. However, one order of magnitude less DNA could still be detected by a random-primed 32P-labelled probe. The ability of nonradioactive and radioactive probes to detect 590-bp gag gene-specific target sequences generated by the polymerase chain reaction (PCR)-mediated amplification of HIV-1 DNA was also compared. Analysis of 20 samples from individuals at increased risk for HIV infection by using the two assayed systems produced virtually equivalent signal images on corresponding specimens. Furthermore, complete concordance in the performance was found when HIV-1 proviral DNA was investigated by PCR in additional 50 samples of human blood mononuclear cells.
Zachar
V
V
Danish Cancer Society, Department of Virus and Cancer, Aarhus.
Mayer
V
V
Aboagye-Mathiesen
G
G
Nørskov-Lauritsen
N
N
Ebbesen
P
P
D000218:Adamantane / Q000031:analogs & derivatives; D001483:Base Sequence; D015139:Blotting, Southern; D004247:DNA; D004279:DNA, Viral / Q000032:analysis*; D015497:HIV-1 / Q000302:isolation & purification*; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D009943:Organophosphorus Compounds; D010761:Phosphorus Radioisotopes; D016133:Polymerase Chain Reaction / Q000379:methods*
D004279:DNA, Viral; D009943:Organophosphorus Compounds; D010761:Phosphorus Radioisotopes; C072934:adamantyl-1,2-dioxetane phosphate; D004247:DNA; D000218:Adamantane