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

被引：59
作者：
Gallina, L.
Dal Pozzo, F.
Mc Innes, C. J.
Cardeti, G.
Guercio, A.
Battilani, M.
Ciulli, S.
Scagliarini, A.
机构：
[1] Alma Mater Studiorum, Dipartimento Sanita Pubbl Vet & Patol Anim, I-40064 Bologna, Italy
[2] Moredun Res Inst, Edinburgh EH26 0PZ, Midlothian, Scotland
[3] Ist Zooprofilattico Sperimentale Lazio & Toscana, I-00178 Rome, Italy
[4] Ist Zooprofilattico Sperimentale Sicilia, I-90129 Palermo, Italy
来源：
JOURNAL OF VIROLOGICAL METHODS | 2006年 / 134卷 / 1-2期
关键词：
ORFV; titre determination; real time PCR; TaqMan (R) PCR;
D O I：
10.1016/j.jviromet.2005.12.014
中图分类号：
Q5 [生物化学];
学科分类号：
071010 ; 081704 ;
摘要：
A real time quantitative PCR assay based on TaqMan (R) 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 (R) 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 x 10(1) to 1 x 10(6) TCID50/ml. A good correlation between the titre determined by the TaqMan (R) 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. (c) 2005 Elsevier B.V. All rights reserved.
引用
收藏
页码：140 / 145
页数：6
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