Microplate capture hybridization of amplified parvovirus B19 DNA fragment labelled with digoxigenin.
A capture hybridization technique in microplate has been developed for the identification of polymerase chain reaction (PCR) amplified B19 DNA fragment in clinical specimens. The amplified 104 bp B19 DNA fragment, located in the gene coding for structural proteins, was directly labelled during the amplification reaction by incorporation of digoxigenin-labelled dUTP. The amplified product was then captured by a probe immobilized on microplate wells. The capture hybridization reaction was visualized as an enzyme-linked immunosorbent assay using anti-digoxigenin Fab fragment labelled with peroxidase. Thirty-five serum samples were tested by our capture hybridization assay and the results were in accordance with the results obtained by Southern blot analysis of PCR amplified product. Our microplate capture hybridization assay showed a high sensitivity and reproducibility and appears to be a practical and reliable test for routine screening of B19 parvovirus DNA in clinical specimens.
Gibellini
D
D
Institute of Microbiology, University of Bologna, Italy.
Zerbini
M
M
Musiani
M
M
Venturoli
S
S
Gentilomi
G
G
La Placa
M
M
D001483:Base Sequence; D015139:Blotting, Southern; D017931:DNA Primers; D004279:DNA, Viral / Q000097:blood* / Q000235:genetics; D004076:Digoxigenin; D005814:Genes, Viral; D006801:Humans; D016867:Immunocompromised Host; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization / Q000295:instrumentation; D016732:Parvovirus B19, Human / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000295:instrumentation; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D015678:Viral Structural Proteins / Q000235:genetics
D017931:DNA Primers; D004279:DNA, Viral; D015678:Viral Structural Proteins; D004076:Digoxigenin