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Selected Publication:

Publication type: Journal Article
Document type: Full Paper

Year: 2005

Author(s): Revilla-Fernández, S; Wallner, B; Truschner, K; Benczak, A; Brem, G; Schmoll, F; Mueller, M; Steinborn, R

Title: The use of endogenous and exogenous reference RNAs for qualitative and quantitative detection of PRRSV in porcine semen.

Source: J Virol Methods. 2005; 126(1-2):21-30



Authors Vetmeduni Vienna:

Brem Gottfried
Müller Mathias
Schmoll Friedrich
Steinborn Ralf
Wallner Barbara

Vetmed Research Units
Institute of Animal Breeding and Genetics
University Clinic for Swine


Abstract:
Semen is known to be a route of porcine reproductive and respiratory syndrome virus (PRRSV) transmission. A method was developed for qualitative and quantitative detection of the seminal cell-associated PRRSV RNA in relation to endogenous and exogenous reference RNAs. As endogenous control for one-step real-time reverse transcription (RT)-PCR UBE2D2 mRNA was selected. Particularly for the analysis of persistent infections associated with low copy numbers of PRRSV RNA, UBE2D2 mRNA is an ideal control due to its low expression in seminal cells and its detection in all samples analysed (n = 36). However, the amount of UBE2D2 mRNA in porcine semen varied (up to 106-fold), thus its use is limited to qualitative detection of PRRSV RNA. For quantitation, a synthetic, non-metazoan RNA was added to the RNA isolation reaction at an exact copy number. The photosynthesis gene ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) from Arabidopsis thaliana was used as an exogenous spike. Unexpectedly, PRRSV RNA was detected in a herd of specific pathogen-free (SPF) boars which were tested ELISA-negative for anti-PRRSV antibodies. Therefore, RT-PCR for seminal cell-associated PRRSV is a powerful tool for managing the SPF status during quarantine programs and for routine outbreak investigations.

Keywords Pubmed: Animals
Molecular Sequence Data
Porcine Reproductive and Respiratory Syndrome/diagnosis*
Porcine Reproductive and Respiratory Syndrome/virology
Porcine respiratory and reproductive syndrome virus/genetics
Porcine respiratory and reproductive syndrome virus/isolation & purification*
RNA, Messenger/analysis
RNA, Viral/analysis*
RNA, Viral/genetics
Reference Standards
Reverse Transcriptase Polymerase Chain Reaction/standards*
Ribulose-Bisphosphate Carboxylase/genetics
Semen/virology*
Sensitivity and Specificity
Sequence Analysis, DNA
Swine
Ubiquitin-Conjugating Enzymes/genetics


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