Microplate Hybridization Research Articles

Microplate hybridization of amplified viral DNA segment

We have developed a simple hybridization method for a DNA segment which is amplified by the polymerase chain reaction: after heat denaturation, the amplified DNA segment with a length of more than 300 bases is adsorbed to microplate wells in the presence of 1.5 M NaCl or 0.5 M ammonium sulfate; the immobilized DNA is hybridized with a biotin-labeled DNA probe; then, the hybridization signal is detected by streptavidin-conjugated beta-galactosidase or peroxidase. This method has several advantages over the conventional dot blot hybridization method: (i) radioisotopes are not used, (ii) synthetic oligonucleotide for the probe is not needed, (iii) the time required for washing of the solid phase is greatly reduced, and (iv) the baking and prehybridization procedures are eliminated. By this method, we were able to detect viral genomes in vesicle specimens from patients infected with varicella-zoster virus.

Evaluation of the microplate hybridization method for rapid identification ofLegionella species

Rapid genetic identification of 23 Legionella species was performed using microdilution plate hybridization. When an isolate appeared to be a strain of the Legionella species, its DNA was quickly extracted, labelled with photobiotin and distributed to microdilution wells where 23 different DNAs had previously been immobilized. After hybridization for 90 min at 45 degrees C, the microdilution well which emitted the maximum fluorescence was determined. The criterion for interpreting results of this genetic identification method is given.

Fertility-associated Λ2-microglobulin (AMGF) as a possible marker of external genital endometriosis

A highly sensitive and nonradioactive microplate hybridization assay for the detection of Epstein–Barr virus (EBV)-specific polymerase chain reaction (PCR) product was developed. The PCR product is labelled by adding digoxigenin-dUTP directly to the reaction mixture and, after denaturation, is captured by a microtitre plate coated with an extravidin-linked biotinylated probe. Captured products are reacted with a peroxidase-conjugated anti-digoxigenin antibody and detected using tetramethylbenzidine. The assay detected less than ten EBV genomes in a background EBV-negative DNA of 0.75 μg and, when tested on clinical samples, it was able to define the viral load in throat washings of patients with acute infectious mononucleosis, immunosuppressed patients with HIV infection, and rare normal individuals who shed the virus in the oropharynx.