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 AD169 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 O 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.
McKeating
J A
JA
Al-Nakib
W
W
Greenaway
P J
PJ
Griffiths
P D
PD
D001710:Biotin; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000235:genetics; D004262:DNA Restriction Enzymes; D004279:DNA, Viral / Q000032:analysis*; D015247:Deoxyribonuclease HindIII; D005347:Fibroblasts; D006801:Humans; D009693:Nucleic Acid Hybridization; D010761:Phosphorus Radioisotopes
D004279:DNA, Viral; D010761:Phosphorus Radioisotopes; D001710:Biotin; D004262:DNA Restriction Enzymes; D015247:Deoxyribonuclease HindIII