Detection and variant identification of HHV-6 by a non-radioactive hybridization microplate assay for amplimers detection.
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.
Gautheret
A
A
Laboratoire de Bactériologie-Virologie, CNRS EP 57, C.E.R.V.I., Groupe Hospitalier Pitié-Salpêtrière, Paris, France.
Aubin
J T
JT
Poirel
L
L
Chopineau
S
S
Agut
H
H
Dussaix
E
E
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*
D017931:DNA Primers; D004279:DNA, Viral; D001710:Biotin