Detection of infectious laryngotracheitis virus infected cells with cloned DNA probes.
A genomic library of infectious laryngotracheitis virus (ILTV) DNA BamH1 fragments was prepared and two cloned fragments were evaluated for their potential as probes for the detection of ILTV infected cells. The virus was purified by a modified sucrose density gradient procedure for the isolation of pure ILTV DNA. A genomic library was constructed using BamH1-digested ILTV DNA and pGEM7 as a vector. A 1.1 kb cloned BamH1 fragment of ILTV DNA was tested in a slot or dot blot assay for the detection of ILTV infected cells. The limit of detection for this probe was at least 0.12 ng of pure ILTV DNA. The probe was able to identify both chicken embryo liver (CELi) cells and choriallantoic membranes infected with ILTV. Chicken embryo liver cells infected with several field isolates and a vaccine strain of ILTV were positive by dot blot analysis using this probe. Some qualitative differences in the degree of hybridization to cells infected by different ILTV isolates were observed. Uninfected cells and cells infected with fowlpox virus, turkey herpesvirus, Marek's disease virus or Newcastle disease virus were negative by the same assay. Compared with the 1.1 kb fragment, a larger 6 kb cloned BamH1 fragment of ILTV DNA showed a stronger hybridization signal to DNA from ILTV infected cells.
Nagy
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Department of Veterinary Microbiology and Immunology, Ontario Verterinary College, University of Guelph.
D000818:Animals; D015139:Blotting, Southern; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D002642:Chick Embryo; D003001:Cloning, Molecular; D015342:DNA Probes; D004279:DNA, Viral / Q000032:analysis* / Q000302:isolation & purification; D004587:Electrophoresis, Agar Gel; D015723:Gene Library; D001352:Herpesvirus 1, Gallid / Q000235:genetics / Q000302:isolation & purification* / Q000648:ultrastructure; D008099:Liver / Q000196:embryology; D008854:Microscopy, Electron; D009693:Nucleic Acid Hybridization; D011237:Predictive Value of Tests; D015183:Restriction Mapping
D015342:DNA Probes; D004279:DNA, Viral