title: Detection and variant identification of HHV-6 by a non-radioactive hybridization microplate assay for amplimers detection.
abstract: A non-radioactive hybridization microtiter plate assay was developed and evaluated for detection of the HHV-6 genome and to identify HHV-6 variants A and B. The viral DNA is amplified by the polymerase chain reaction using a 5'-end-biotinylated primer. The biotinylated amplimers are captured on avidin-coated microtiter plates, denaturated with sodium hydroxide and hybridized to a 3'-end-digoxigenin-labelled probe. Subsequently, anti-digoxigenin Fab fragments conjugated with alkaline phosphatase are used for the revelation of the hybridized probe. The result is obtained by measuring the intensity of light emitted with a spectrophotometer. This new assay was compared to the standard analysis of amplified products by Southern hybridization consisting of gel electrophoresis of the amplimers, transfer onto a nylon membrane, and hybridization with a 32P-labelled oligomeric probe. Both methods exhibited the same sensitivity and specificity. Thus, a non-radioactive hybridization microtiter plate assay may be a suitable alternative to isotopic techniques.
authors.affiliation: Laboratoire de Bactériologie-Virologie, CNRS EP 57, C.E.R.V.I., Groupe Hospitalier Pitié-Salpêtrière, Paris, France.
authors.forename: A
authors.initials: A
authors.lastname: Gautheret
authors.forename: J T
authors.initials: JT
authors.lastname: Aubin
authors.forename: L
authors.initials: L
authors.lastname: Poirel
authors.forename: S
authors.initials: S
authors.lastname: Chopineau
authors.forename: H
authors.initials: H
authors.lastname: Agut
authors.forename: E
authors.initials: E
authors.lastname: Dussaix
mesh_terms: D001710:Biotin; D015139:Blotting, Southern; D002478:Cells, Cultured; D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis; D014644:Genetic Variation; D006566:Herpesviridae Infections / Q000473:pathology / Q000821:virology*; D015654:Herpesvirus 6, Human / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods*
chemical_list: D017931:DNA Primers; D004279:DNA, Viral; D001710:Biotin