Heminested PCR assay for detection of six genotypes of rabies and rabies-related viruses.
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.
Heaton
P R
PR
Department of Virology, Veterinary Laboratories Agency, Addlestone, Surrey, United Kingdom. pheaton@hgmp.mrc.ac.uk
Johnstone
P
P
McElhinney
L M
LM
Cowley
R
R
O'Sullivan
E
E
Whitby
J E
JE
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
D017931:DNA Primers