Detection of an arbovirus in an invertebrate and a vertebrate host using the polymerase chain reaction.
The ability of the polymerase chain reaction (PCR) to diagnose an arboviral infection in an arthropod vector or a mammalian host was examined. Dugbe (DUG) viral RNA was detected in RNA extracts from infected tissue samples by reverse transcription and enzymatic amplification of the resulting cDNA using Taq DNA polymerase, followed by characterisation of the amplified product by agarose gel electrophoresis or dot-blot hybridisation. Viral RNA was detected in the organs and haemolymph of infected Amblyomma variegatum ticks, and in the brain and blood of infected mice. The PCR technique was found to be as sensitive as a plaque assay for detecting DUG virus, but not as sensitive as intracerebral inoculation of mice. The sensitivity of the technique was greatest using crude RNA extracts combined with dot-blot analysis of the resulting PCR products using a DUG specific cDNA probe. A result was obtained within 48 h using PCR whereas biological assays took at least 8 days to diagnose the virus infection.
Ward
V K
VK
NERC Institute of Virology and Environmental Microbiology, Oxford, U.K.
Marriott
A C
AC
Booth
T F
TF
el-Ghorr
A A
AA
Nuttall
P A
PA
D000818:Animals; D001102:Arbovirus Infections / Q000097:blood / Q000175:diagnosis*; D001103:Arboviruses / Q000235:genetics* / Q000472:pathogenicity; D001483:Base Sequence; D001921:Brain / Q000382:microbiology; D002478:Cells, Cultured; D004259:DNA-Directed DNA Polymerase; D006458:Hemolymph / Q000382:microbiology; D051379:Mice; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012367:RNA, Viral / Q000032:analysis*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D015183:Restriction Mapping; D019914:Taq Polymerase; D013987:Ticks / Q000382:microbiology*; D014774:Virulence
D012367:RNA, Viral; D019914:Taq Polymerase; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase