An improved dot-blot method for virus detection in chicken embryo fibroblast cultures.
A simplified dot-blot procedure is described for the detection of fowlpox virus (FPV) in infected monolayers of chicken embryo fibroblasts (CEF) cultured in 96-well microtiter plates. The relative resistance of DNA to hot NaOH, which hydrolyzes other macromolecules including RNA and protein, was exploited to solubilize virus infected cells and denature intracellular DNA in a simple, quick manner. Moreover, there was no need to purify virus or isolate viral DNA from cellular DNA prior to dot blotting. After incubation of CEF with FPV, the extracellular fluid from infected cells was collected for storage in 96-well microtiter plates. The remaining cell monolayers in each well were then solubilized with hot NaOH. The solubilized and denatured DNA was transferred to a nylon membrane using a dot-blot vacuum filtration manifold. Hybridization was carried out with a 32P-labeled FPV DNA probe. With this methodology it was possible to detect specific viral DNA sequences following the infection of cell monolayers with as little as 1 infectious unit per well. The ability to detect specific viral DNA sequences in infected cells, without the need to isolate pure viral DNA, made it possible to analyze large numbers of samples in a single experiment. Moreover, sufficient fowlpox virus was present in the extracellular media from each well for further amplification and analysis of selected samples.
Hammermueller
J D
JD
Department of Veterinary Microbiology, University of Guelph, Ontario, Canada.
Krell
P J
PJ
Derbyshire
J B
JB
Nagy
E
E
D000818:Animals; D002478:Cells, Cultured; D002642:Chick Embryo; D004279:DNA, Viral / Q000032:analysis*; D005347:Fibroblasts / Q000382:microbiology*; D005587:Fowlpox virus / Q000235:genetics / Q000302:isolation & purification*; D006868:Hydrolysis; D008722:Methods; D009693:Nucleic Acid Hybridization; D012680:Sensitivity and Specificity; D012972:Sodium Hydroxide
D004279:DNA, Viral; D012972:Sodium Hydroxide