Detection of cytomegalovirus by dot-blot DNA hybridization using probes labelled with 32P by nick translation or random hexanucleotide priming.
A DNA hybridization assay for the detection of human cytomegalovirus (HCMV) DNA was developed using random hexanucleotide-primed 32P-labelled Hind III restriction fragments of HCMV DNA as probes, and compared with a DNA hybridization assay using probes labelled with 32P by nick translation. Nick-translated probes were shown to be able to detect between 1 and 10 pg of homologous DNA or the DNA of 10-50 HCMV-infected fibroblasts. Random hexanucleotide-primed DNA probes lowered these detection limits to 0.1-0.5 pg of homologous DNA or one to five HCMV-infected fibroblasts. An increase in the autoradiographic exposure time from 18 h to 4 days increased the level of detection for homologous DNA or HCMV-infected fibroblast DNA by approximately five-fold. Preliminary screening of 35 urine samples by DNA hybridization using a random hexanucleotide-primed probe correctly identified three samples positive by virus isolation in tissue culture or immediate-early nuclear antigen detection and 29 of 32 samples negative by tissue culture.
Kimpton
C P
CP
Department of Medical Microbiology, University of Manchester, UK.
Corbitt
G
G
Morris
D J
DJ
D001345:Autoradiography / Q000379:methods; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000235:genetics*; D015342:DNA Probes; D004279:DNA, Viral / Q000235:genetics*; D014645:Herpesvirus 3, Human / Q000235:genetics*; D006801:Humans; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes; D010761:Phosphorus Radioisotopes; D014176:Protein Biosynthesis; D018139:Simplexvirus / Q000235:genetics*
D015342:DNA Probes; D004279:DNA, Viral; D015345:Oligonucleotide Probes; D010761:Phosphorus Radioisotopes