Rapid identification of bluetongue virus by nucleic acid hybridization in solution

Charles A. Dangler, Stephen J. Dunn, Kevin R E Squire, Jeffrey L Stott, Bennie Osburn

Research output: Contribution to journalArticle

16 Scopus citations

Abstract

A liquid phase hybridization format has been adapted for the identification of bluetongue virus (BTV) with respect to serogroup and serotype. In this paper we present experiments which demonstrate the identification of bluetongue virus with respect to both serogroup and serotype. BTV serogroup-specific and BTV serotype 17-specific single-stranded RNA probes labeled with 35S-CTP were used to identify BTV viral nucleic acids in infected cell culture lysates. Cell lysates were prepared for testing by rapid solubilization in a guanidine isothiocyanate/EDTA/dithiothreitol medium. Hybridizations were done in 12.5 μl reaction volumes for 2 h or overnight using nanogram quantities of probe. An RNAse solution was added subsequent to hybridization and incubated for 30 min. TCA-precipitable counts were collected and quantitated by scintillation counting. Specific hybridization signals were obtained in as little as 3 h in assays consisting of uninfected controls, cells infected with different BTV serotypes, and cells infected with a strain of epizootic hemorrhagic disease of deer (EHDV-1). By using asymmetric single-stranded RNA probes we were able to distinguish viral mRNA and viral double-stranded RNA components. We propose this technique as an ancillary or replacement procedure for the confirmation and classification of viral isolates using an appropriate battery of nucleic acid probes. Potential applications and methods of enhancing the technique are discussed.

Original languageEnglish (US)
Pages (from-to)353-365
Number of pages13
JournalJournal of Virological Methods
Volume20
Issue number4
DOIs
StatePublished - 1988

Keywords

  • Bluetongue virus
  • Diagnostics
  • Solution hybridization

ASJC Scopus subject areas

  • Virology

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