title: TaqMan real-time PCR assay based on DNA polymerase gene for rapid detection of Orf infection.
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.
authors.affiliation: Pox Virus Laboratory, Division of Virology, Indian Veterinary Research Institute, Nainital (Distt.), Mukteswar 263 138, Uttarakhand, India.
authors.forename: D P
authors.initials: DP
authors.lastname: Bora
authors.forename: G
authors.initials: G
authors.lastname: Venkatesan
authors.forename: V
authors.initials: V
authors.lastname: Bhanuprakash
authors.forename: V
authors.initials: V
authors.lastname: Balamurugan
authors.forename: M
authors.initials: M
authors.lastname: Prabhu
authors.forename: M S
authors.initials: MS
authors.lastname: Siva Sankar
authors.forename: R
authors.initials: R
authors.lastname: Yogisharadhya
mesh_terms: D000818:Animals; D004259:DNA-Directed DNA Polymerase / Q000235:genetics; D004474:Ecthyma, Contagious / Q000175:diagnosis* / Q000821:virology; D006041:Goats; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009923:Orf virus / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012756:Sheep; D013997:Time Factors; D014730:Veterinary Medicine / Q000379:methods*; D019562:Viral Load / Q000379:methods; D014764:Viral Proteins / Q000235:genetics; D014773:Virology / Q000379:methods*
chemical_list: D014764:Viral Proteins; D004259:DNA-Directed DNA Polymerase