[Use of ultrastructural in situ hybridization for the intracellular detection of viral DNA and RNA].
Post-embedding in situ nucleic acid hybridization (ISH) at the electron microscope level is at present the best tool for the ultrastructural detection of specific viral sequences in infected cells since sensitivity and specificity of the method are paralleled by preservation of ultrastructure. Biotin-labelled double-stranded viral DNA probes were applied at the surface of lowicryl K4M sections for localizing viral nucleic acids (all viral DNA, only single-stranded viral DNA, or viral RNA) in herpes simplex type 1 and adenovirus type 5 infected cells. The hybrids were revealed by immunocytological technique using gold particles as marker. Our data demonstrate that several biological characteristics of the infected cells could lead to false negative or positive results. Therefore, total elimination of artifacts by appropriate experimental conditions is required for the identification of defined viral nucleic acids sequences in the nucleoprotein complexes.
Puvion-Dutilleul
F
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Laboratoire de Biologie et Ultrastructure du Noyau de l'UPR 272 du CNRS, Villejuif, France.
D000180:Acrylic Resins; D000260:Adenoviruses, Human / Q000235:genetics*; D000818:Animals; D004279:DNA, Viral / Q000032:analysis* / Q000648:ultrastructure; D017403:In Situ Hybridization / Q000379:methods*; D066298:In Vitro Techniques; D008854:Microscopy, Electron; D012367:RNA, Viral / Q000032:analysis* / Q000648:ultrastructure; D011817:Rabbits; D018139:Simplexvirus / Q000235:genetics*
D000180:Acrylic Resins; D004279:DNA, Viral; D012367:RNA, Viral; C037883:Lowicryl 4KM resin