Molecular analysis of contagious pustular dermatitis virus: a simplified method for viral DNA extraction from scab material.
A new simplified method of DNA extraction of contagious pustular dermatitis virus directly from scab material of natural and experimental infections is described. Scabs are suspended in buffer solution and an enriched core suspension is obtained after treatment with detergent, quelants and centrifugation. DNA is isolated after proteinase digestion and phenolchloroform extraction. Viral DNA and fragments with sizes ranging from 23-25 kb were observed by agarose gel electrophoresis. This DNA was used for digestion with several restriction endonucleases which produced parapoxvirus-specific patterns. Southern blots with the TK gene of vaccinia virus as probe confirmed the virus as being poxvirus-related and allowed a preliminary TK gene location for our isolates. The method was developed in order to allow a quick epidemiological survey of contagious pustular dermatitis virus in Brazil, eliminating the need for time-consuming and expensive viral propagation in cell culture and purification.
Mazur
C
C
Instituto de Veterinária, Universidade Federal Rural do Rio Janeiro UFRRJ, Itaguaí, Brazil.
Rangel Filho
F B
FB
Galler
R
R
D000818:Animals; D016172:DNA Fingerprinting; D015342:DNA Probes; D004279:DNA, Viral / Q000302:isolation & purification* / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D015511:Goat Diseases / Q000382:microbiology*; D006041:Goats; D009923:Orf virus / Q000235:genetics*; D014616:Vaccinia virus / Q000235:genetics
D015342:DNA Probes; D004279:DNA, Viral; D015252:Deoxyribonucleases, Type II Site-Specific