Detection of cytomegalovirus by DNA-DNA hybridization employing probes labelled with 32-phosphorus or biotin

Various factors influencing the detection of human cytomegalovirus (HCMV) in infected cells by DNA-DNA hybridization have been investigated. Employing the Hind III O fragment of HCMV AD 169 labelled with ³²P, we found that detection sensitivity was highly influenced by the method employed for extraction of DNA from infected cells. Excision of the Hind III 0 fragment from the vector by restriction endonuclease digestion prior to ³²P-labelling further improved the detection capability of the probe. Similarly, cytomegalovirus (CMV) DNA detection employing biotin-labelled probes and streptavidin/alkaline phosphatase in the hybridot assay was also highly dependent on the method of DNA extraction prior to hybridization. Finally, we describe an in situ assay employing a biotin-labelled probe and fluorescein-conjugated avidin to detect CMV DNA in cultured cells.