Development and optimisation of a duplex real-time reverse transcription quantitative PCR assay targeting the VP7 and NS2 genes of African horse sickness virus

M. Quan, C. W. Lourens, Nigel J Maclachlan, Ian Gardner, A. J. Guthrie

Research output: Contribution to journalArticle

26 Scopus citations

Abstract

Nucleotide sequences of 52 South African isolates of African horse sickness virus (AHSV) collected during 2004-2005 and including viruses of all nine AHSV serotypes, were used to design and develop a duplex real-time reverse transcription quantitative PCR (RT-PCR) assay targeting the VP7 (S8) and NS2 (S9) genes of AHSV. The assay was optimized for detection of AHSV in fresh and frozen blood of naturally infected horses. Assay performance was enhanced using random hexamers rather than gene-specific primers for RT, and with denaturation of double-stranded RNA in the presence of random hexamers. The assay was efficient with a linear range of at least five orders of magnitude. The analytical sensitivity of the assay was 132 copies of the target genes (4125 copies per ml of blood), and the assay was at least 10-fold more sensitive than virus isolation on BHK-21 cells. The assay was also highly specific because it did not detect related orbiviruses, such as bluetongue and equine encephalosis viruses.

Original languageEnglish (US)
Pages (from-to)45-52
Number of pages8
JournalJournal of Virological Methods
Volume167
Issue number1
DOIs
StatePublished - Jul 2010

Keywords

  • African horse sickness virus
  • Duplex
  • NS2 gene
  • Real-time quantitative RT-PCR
  • VP7 gene

ASJC Scopus subject areas

  • Virology

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