Whole-genome analysis of pseudorabies virus gene expression by real-time quantitative RT-PCR assay

Abstract

Background: Pseudorabies virus (PRV), a neurotropic herpesvirus of pigs, serves as an excellent model system with which to investigate the herpesvirus life cycle both in cultured cells and in vivo. Real-time RT-PCR is a very sensitive, accurate and reproducible technique that can be used to detect very small amounts of RNA molecules, and it can therefore be applied for analysis of the expression of herpesvirus genes from the very early period of infection.

Analysis of rice stripe virus whole-gene expression in rice and in the small brown planthopper by real-time quantitative PCR

“…Additionally, this technology has been proved to be effi cient for analysis of virus gene expression, for example, Eepstein-Barr virus (Pan et al, 2005) and pseudorabies virus (Tombacz et al, 2009). Zhang et al (2008a) reported one-step real time RT-PCR methods for quantifying RSV-CP gene in rice tissues and in SBPH.…”

Effect of us1 gene mutation on pseudorabies virus gene expression

“…Us1(ICP22 in HSV) is also a regulatory gene, which is an odd-one-out, becausebased on previous experiments there is no consensus as to whether the PRV us1 gene is expressed in IE, E or L kinetics [27][28][29]. Interestingly, us1, similar to ie180 is located in the IR region of the genome; therefore PRV has two copies of us1 genes so as of ie180 gene.…”

“…Primers pairs were used as described previously [29]. Briefly, primer pairs was designed using the Primer Express program (Applied Biosystems) and the FastPCR Professional …”

Analysis of mutant Pseudorabies virus strains

“…KG), as described previously [21]. Briefly, after the cells had been collected by centrifugation and lysed by buffer containing chaotropic ions, the nucleic acids were docked to a silica column.…”

“…H 2 O was included as a no-template control, and cDNA derived from the reverse-transcribed RNAs of non-infected cells was used as a negative mock-infected control. We applied SYBR Green-based real-time PCR because of the lower costs and simpler protocol than for TaqMan probe-based methods for instance [21]. It has recently been demonstrated that the SYBR-based method of detection is as sensitive and specific, and has a similar dynamic range to that of the TaqMan-based technique [81].…”