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 log10 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 Å~ 101 to 1 Å~ 106 TCID50/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.

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

GALLINA, LAURA;DAL POZZO, FABIANA;BATTILANI, MARA;CIULLI, SARA;SCAGLIARINI, ALESSANDRA
2006

Abstract

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 log10 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 Å~ 101 to 1 Å~ 106 TCID50/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.
2006
L. Gallina; F. Dal Pozzo; C.J. Mc Innes; G. Cardeti; A. Guercio; M. Battilani; S. Ciulli; A. Scagliarini
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/23191
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