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 Citations (Scopus)

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

Fingerprint

Bluetongue virus
Nucleic Acid Hybridization
RNA Probes
Nucleic Acid Probes
Cytidine Triphosphate
Scintillation Counting
Deer
Double-Stranded RNA
Dithiothreitol
Viral RNA
Serogroup
Edetic Acid
Nucleic Acids
Cell Culture Techniques
Messenger RNA

Keywords

  • Bluetongue virus
  • Diagnostics
  • Solution hybridization

ASJC Scopus subject areas

  • Virology

Cite this

Rapid identification of bluetongue virus by nucleic acid hybridization in solution. / Dangler, Charles A.; Dunn, Stephen J.; Squire, Kevin R E; Stott, Jeffrey L; Osburn, Bennie.

In: Journal of Virological Methods, Vol. 20, No. 4, 1988, p. 353-365.

Research output: Contribution to journalArticle

Dangler, Charles A. ; Dunn, Stephen J. ; Squire, Kevin R E ; Stott, Jeffrey L ; Osburn, Bennie. / Rapid identification of bluetongue virus by nucleic acid hybridization in solution. In: Journal of Virological Methods. 1988 ; Vol. 20, No. 4. pp. 353-365.
@article{0db439ff1de94c9bb076ddca14d8bcb2,
title = "Rapid identification of bluetongue virus by nucleic acid hybridization in solution",
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.",
keywords = "Bluetongue virus, Diagnostics, Solution hybridization",
author = "Dangler, {Charles A.} and Dunn, {Stephen J.} and Squire, {Kevin R E} and Stott, {Jeffrey L} and Bennie Osburn",
year = "1988",
doi = "10.1016/0166-0934(88)90138-3",
language = "English (US)",
volume = "20",
pages = "353--365",
journal = "Journal of Virological Methods",
issn = "0166-0934",
publisher = "Elsevier",
number = "4",

}

TY - JOUR

T1 - Rapid identification of bluetongue virus by nucleic acid hybridization in solution

AU - Dangler, Charles A.

AU - Dunn, Stephen J.

AU - Squire, Kevin R E

AU - Stott, Jeffrey L

AU - Osburn, Bennie

PY - 1988

Y1 - 1988

N2 - 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.

AB - 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.

KW - Bluetongue virus

KW - Diagnostics

KW - Solution hybridization

UR - http://www.scopus.com/inward/record.url?scp=0024061526&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0024061526&partnerID=8YFLogxK

U2 - 10.1016/0166-0934(88)90138-3

DO - 10.1016/0166-0934(88)90138-3

M3 - Article

C2 - 2846603

AN - SCOPUS:0024061526

VL - 20

SP - 353

EP - 365

JO - Journal of Virological Methods

JF - Journal of Virological Methods

SN - 0166-0934

IS - 4

ER -