Bluetongue virus detection: a safer reverse-transcriptase polymerase chain reaction for prediction of viremia in sheep.
A reversible target capture viral RNA extraction procedure was combined with a reverse-transcriptase nested polymerase chain reaction (PCR) to develop a capture PCR assay providing a rapid and safe prediction method for circulating bluetongue virus in infected ruminants. This new assay was compared with virus isolation and a recently developed antigen-capture enzyme-linked immunosorbent assay (ELISA) for the detection of bluetongue virus. Eight Warhill crossbred sheep were inoculated subcutaneously with bluetongue virus serotype 10, and blood samples were taken sequentially over a period of 28 days. The capture PCR detected the peak of viremia, as determined by virus isolation and antigen-capture ELISA, from day 5 to day 14 after challenge. The results indicate that the rapid-capture bluetongue virus PCR provides a rapid indicator of samples in which virus can be isolated. In addition, this capture bluetongue virus PCR procedure does not require a lengthy phenol extraction or the use of the highly toxic methyl mercury hydroxide denaturant.
Shad
G
G
College of Veterinary Medicine, Washington State University, Pullman, USA.
Wilson
W C
WC
Mecham
J O
JO
Evermann
J F
JF
D000818:Animals; D001819:Bluetongue / Q000175:diagnosis*; D001820:Bluetongue virus / Q000302:isolation & purification*; D017931:DNA Primers; D004198:Disease Susceptibility; D004587:Electrophoresis, Agar Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000097:blood / Q000302:isolation & purification; D012756:Sheep; D014766:Viremia / Q000175:diagnosis*
D017931:DNA Primers; D012367:RNA, Viral