Chemiluminescent microtiter method for detecting PCR amplified HIV-1 DNA.
A rapid and sensitive method for detecting HIV-1 DNA sequences amplified by polymerase chain reaction (PCR) is described. The method uses solution phase hybridization for rapid and efficient annealing between digoxigenin-labeled targets and biotinylated capture probes. Hybrids containing biotin are captured onto streptavidin coated microwells and all other PCR components are washed away, including spurious amplification products. The presence of the digoxigenin-labeled amplified HIV target is then detected by anti-digoxigenin-alkaline phosphatase conjugates using the chemiluminescent substrate PPD. This approach maintains high specificity by nucleic acid dependent capture, and high sensitivity by efficient solution hybridization. The method is rapid (2 hours), and capable of detecting 10 HIV targets.
Suzuki
K
K
Biomedical Research Center, Olympus Corporation, East Setauket, NY 11733.
Okamoto
N
N
Watanabe
S
S
Kano
T
T
D000469:Alkaline Phosphatase; D001426:Bacterial Proteins; D001483:Base Sequence; D001710:Biotin; D004279:DNA, Viral / Q000032:analysis*; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D019809:Streptavidin
D001426:Bacterial Proteins; D004279:DNA, Viral; D001710:Biotin; D019809:Streptavidin; D000469:Alkaline Phosphatase