A real time PCR assay for the detection and quantification of orf virus

• Published in: Journal of virological methods Vol. 134; no. 1; pp. 140 - 145
• Main Authors: Gallina, L., Dal Pozzo, F., Mc Innes, C.J., Cardeti, G., Guercio, A., Battilani, M., Ciulli, S., Scagliarini, A.
• Format: Journal Article
• Language: English
• Published: London Elsevier B.V 01.06.2006; Amsterdam Elsevier; New York, NY
• Subjects: Animals; Biological and medical sciences; Cattle; Cells, Cultured; Coculture Techniques; Ecthyma, Contagious - virology; Fundamental and applied biological sciences. Psychology; Genes, Viral; Goats; Humans; Keratinocytes - virology; Microbiology; Orf virus; Orf virus - genetics; Orf virus - growth & development; Orf virus - isolation & purification; ORFV; Polymerase Chain Reaction - methods; Real time PCR; Reproducibility of Results; Rupicapra; Sheep; Skin; TaqMan ® PCR; Techniques used in virology; Titre determination; Vaccinia virus; Viral Envelope Proteins - genetics; Viral Plaque Assay; Virology; TaqMan ® PCR; Titre determination; Real time PCR; ORFV; Chordopoxvirinae; Microbiology; Method; Orf virus; Real time; Virology; Virus; Polymerase chain reaction; TaqMan® PCR; Poxviridae; Detection; Parapoxvirus; Quantitative analysis
• ISSN: 0166-0934; 1879-0984
• DOI: 10.1016/j.jviromet.2005.12.014

A real time quantitative PCR assay based on TaqMan ® technology was developed for orf virus (ORFV) DNA quantification in clinical samples, infected cells and organotypic cultures. This method was based on the amplification of a 70 bp fragment from the ORFV B2L gene (orthologue of the Vaccinia virus Copenhagen F13L gene) that encodes the major envelope protein. Both intra- and inter-assay variability were well within ±0.25 log 10 S.D. showing the high efficiency and reproducibility of the assay. The TaqMan ® PCR was subsequently used to determine the titre of several batches of the ORFV strain NZ-2, with it being possible to quantify virus solutions in the range of 1 × 10 1 to 1 × 10 6 TCID 50/ml. A good correlation between the titre determined by the TaqMan ® PCR and by conventional endpoint dilution was found. The PCR assay is reproducible and can be used for a rapid quantification of ORFV in vitro and ex vivo, being readily achievable within 1 h.