Detection and variant identification of HHV-6 by a non-radioactive hybridization microplate assay for amplimers...

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.