A multiplex PCR for simultaneous detection and differentiation of North American serotypes of bluetongue and epizootic hemorrhagic disease viruses

Imadeldin E. Aradaib, Wayne L. Smith, Bennie Osburn, James S Cullor

Research output: Contribution to journalArticle

45 Citations (Scopus)

Abstract

In the present study, a multiplex RT-PCR-based assay for simultaneous detection and differentiation of North American serotypes of bluetongue (BT) virus (BTV) and epizootic hemorrhagic disease (EHD) virus (EHDV) in cell culture and clinical samples was developed. Two pairs of primers (B1 and B4) and (E1 and E4) were designed to hybridize to non-structural protein 1 (NS1) genomes of (BTV-11) and (EHDV-1), respectively. The multiplex PCR-based assay utilized a single tube-PCR amplification in which EHDV and BTV primers were used simultaneously in a multiplex format. The BTV primers generated a 790 base pair (bp) specific PCR product from RNA samples of North American BTV serotypes 2, 10, 11, 13 and 17; whereas EHDV serotypes 1 and 2 or total nucleic acid extract from non-infected baby hamster kidney (BHK) cells failed to demonstrate the 790 bp specific BTV PCR product. Likewise, the EHDV primers produced a 387 bp specific PCR product from RNA samples of EHDV serotypes 1 and 2, but not from BTV serotypes 2, 10, 11, 13, 17 or from total nucleic acid extract of BHK cell controls. Two pairs of nested primers (B2 and B3) and (E2 and E3), internal to the annealing sites of primers (B1 and B4) and primers (E1 and E4), produced a 520 bp specific BTV and a 224 bp specific EHDV PCR product from BTV and EHDV first amplification products, respectively. These nested amplifications increased the sensitivity of the PCR assay and confirmed the specificity of the first amplified EHDV or BTV PCR products. The described multiplex RT-PCR-based assay could be used to facilitate rapid detection and differentiation of North American BTV and EHDV serotypes and to provide a valuable tool to study the epidemiology of these orbivirus infections in susceptible animal populations.

Original languageEnglish (US)
Pages (from-to)77-87
Number of pages11
JournalComparative Immunology, Microbiology and Infectious Diseases
Volume26
Issue number2
DOIs
StatePublished - Mar 2003

Fingerprint

Epizootic hemorrhagic disease virus
Bluetongue
bluetongue
Multiplex Polymerase Chain Reaction
serotypes
Viruses
viruses
Base Pairing
Polymerase Chain Reaction
Serogroup
assays
kidney cells
hamsters
Cricetinae
Nucleic Acids
nucleic acids

Keywords

  • Diagnosis-viruses
  • Genetics
  • Orbivirus serogroup
  • Serogroup specificity

ASJC Scopus subject areas

  • Animal Science and Zoology
  • Immunology
  • Microbiology
  • veterinary(all)

Cite this

A multiplex PCR for simultaneous detection and differentiation of North American serotypes of bluetongue and epizootic hemorrhagic disease viruses. / Aradaib, Imadeldin E.; Smith, Wayne L.; Osburn, Bennie; Cullor, James S.

In: Comparative Immunology, Microbiology and Infectious Diseases, Vol. 26, No. 2, 03.2003, p. 77-87.

Research output: Contribution to journalArticle

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