title: Heminested PCR assay for detection of six genotypes of rabies and rabies-related viruses.
abstract: A heminested reverse transcriptase PCR (hnRT-PCR) protocol which is rapid and sensitive for the detection of rabies virus and rabies-related viruses is described. Sixty isolates from six of the seven genotypes of rabies and rabies-related viruses were screened successfully by hnRT-PCR and Southern blot hybridization. Of the 60 isolates, 93% (56 of 60) were positive by external PCR, while all isolates were detected by heminested PCR and Southern blot hybridization. We also report on a comparison of the sensitivity of the standard fluorescent-antibody test (FAT) for rabies antigen and that of hnRT-PCR for rabies viral RNA with degraded tissue infected with a genotype 1 virus. Results indicated that FAT failed to detect viral antigen in brain tissue that was incubated at 37 degrees C for greater than 72 h, while hnRT-PCR detected viral RNA in brain tissue that was incubated at 37 degrees C for 360 h.
authors.affiliation: Department of Virology, Veterinary Laboratories Agency, Addlestone, Surrey, United Kingdom. pheaton@hgmp.mrc.ac.uk
authors.forename: P R
authors.initials: PR
authors.lastname: Heaton
authors.forename: P
authors.initials: P
authors.lastname: Johnstone
authors.forename: L M
authors.initials: LM
authors.lastname: McElhinney
authors.forename: R
authors.initials: R
authors.lastname: Cowley
authors.forename: E
authors.initials: E
authors.lastname: O'Sullivan
authors.forename: J E
authors.initials: JE
authors.lastname: Whitby
mesh_terms: D000818:Animals; D000829:Animals, Domestic; D000835:Animals, Wild; D001483:Base Sequence; D017931:DNA Primers; D005838:Genotype; D016133:Polymerase Chain Reaction / Q000379:methods*; D011818:Rabies / Q000175:diagnosis*; D011820:Rabies virus / Q000145:classification / Q000254:growth & development / Q000302:isolation & purification*; D012209:Rhabdoviridae / Q000145:classification / Q000254:growth & development / Q000302:isolation & purification*; D018353:Rhabdoviridae Infections / Q000175:diagnosis / Q000662:veterinary*; D012680:Sensitivity and Specificity; D016415:Sequence Alignment
chemical_list: D017931:DNA Primers