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TaqMan real-time PCR assay based on DNA polymerase gene for rapid detection of Orf infection

Authors
Journal
Journal of Virological Methods
0166-0934
Publisher
Elsevier
Volume
178
Identifiers
DOI: 10.1016/j.jviromet.2011.09.005
Keywords
  • Orf Virus
  • Dna Polymerase Gene
  • Real Time Pcr
  • Taqman Probe
  • Diagnosis
Disciplines
  • Biology
  • Chemistry
  • Medicine

Abstract

Abstract Both conventional and real time PCR (rt-PCR) assays based on the amplification of a 103bp fragment from the DNA polymerase (DNA pol) gene (conserved, non-structural) of Orf virus (ORFV) were developed for detection and semi-quantitation of ORFV DNA from infected cell culture and clinical samples. The latter technique was based on TaqMan chemistry. The rt-PCR assay was specific and sensitive as it could detect as low as 3.5fg or 15 copies of ORFV genomic DNA. Both intra- (0.38–1.0%) and inter-assay (0.53–2.87%) variabilities of rt-PCR were within the acceptable range meaning the high efficiency and reproducibility of the assay. The rt-PCR was applied successfully to detect ORFV DNA from suspected clinical samples. Further, the assay has shown a relative diagnostic sensitivity and specificity of 100% and 93.5%, respectively, when compared to B2L gene based semi-nested PCR implying a wide potential of this rt-PCR for rapid field diagnosis of Orf in sheep and goats.

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