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.
L. Gallina, F. Dal Pozzo, C.J. Mc Innes, G. Cardeti, A. Guercio, M. Battilani, et al. (2006). A real time PCR assay for the detection and quantification of orf virus. JOURNAL OF VIROLOGICAL METHODS, 134, 142-147 [10.1016/j.jviromet.2005.12.014].
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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
