Quantitation of cytomegalovirus DNA by blot hybridization in blood leukocytes of viremic patients.
We describe a technique for quantitation of viral DNA in blood leukocytes during viremic infection with human cytomegalovirus (CMV). Using a cloned subgenomic DNA probe and a blot hybridization assay, small amounts of viral DNA within samples of leukocyte DNA could be quantitated reproducibly using a videodensitometer. Critical components of the assay were: (1) a direct relationship between optical density and known amounts of viral DNA diluted in cellular DNA as positive standard samples and, (2) determination of the proper duration of autoradiographic exposure. The technique was sufficiently sensitive to detect 10 picograms of CMV DNA in the presence of microgram quantities of host cell DNA. In addition, samples containing minute amounts of CMV DNA could reliably be distinguished from samples that were negative. We have used the technique to characterize the pathogenesis of CMV viremia and to monitor the effects of antiviral chemotherapy. This procedure could easily be applied to pathogenetic studies of a variety of other infectious agents.
Saltzman
R L
RL
Metropolitan-Mt. Sinai Medical Center.
Quirk
M R
MR
Mariash
C N
CN
Jordan
M C
MC
D003587:Cytomegalovirus / Q000235:genetics / Q000302:isolation & purification*; D003586:Cytomegalovirus Infections / Q000097:blood*; D015342:DNA Probes; D004279:DNA, Viral / Q000032:analysis*; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D066298:In Vitro Techniques; D007962:Leukocytes / Q000382:microbiology; D009693:Nucleic Acid Hybridization; D012984:Software; D014766:Viremia / Q000097:blood* / Q000382:microbiology
D015342:DNA Probes; D004279:DNA, Viral