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Publication type: Doctoral Thesis

Year: 2002

Author(s): Höller, K

Title: Qualitativer und quantitativer Nachweis von Porzinem Reproduktivem und Respiratorischem Syndrom-Virus (PRRSV) im Serum mittels One-Step Single-Tube Real-Time Reverse Transkription-Polymerasekettenreaktion.

Source: Dissertation, Vet. Med. Univ. Wien, pp. 134.

Advisor(s):

Schuh Maximilian


Project(s): Qualitative and quantitative detection of PRRSV in serum by one-step single-tube real-time RT-PCR


Abstract:
The Porcine Reproductive and Respiratory Syndrome (PRRS) is one of the economically most important infectious diseases of swine (TERPSTRA et al., 1991). The PRRS virus belongs to the family of Arteriviridae and is endemic in almost all pig-producing countries around the world (MEREDITH, 1995). A high mutation rate is characteristic for this pathogen so that many genotypic variations exist. They all belong either to the group of the European or to the group of the North American isolates. The aim of this study was to develop a sensitive and reliable method for the detection, differentiation and quantification of European and North American PRRSV antigen in the highly conserved ORF6 region of the viral genome. Two methods for the isolation of RNA from liquid samples and two commercially available RT-PCR kits were tested. For virus detection one-step single-tube real-time RT-PCR assays with optimal amplification efficiency were established. The assay sensitivity, measured by using an in vitro transcribed RNA, was 61 copies/reaction for North American virus types. For European isolates it was within a comparable range. The present method is the most sensitive PRRSV quantification assay for serum. Relative quantification was performed using an exogenous mRNA. The basics for a relative quantification of the viral load using an endogenous control were created. The housekeeping gene cyclophilin was described in serum for the first time. For GAPDH a minor influence of an infection with PRRSV on the gene expression was shown. Furtheron the method was used for routine diagnosis of 305 samples to proof its suitability in practice. The results obtained have shown that this method tested can be recommended to routinely detect the PRRSV in serum.

Keywords:
PRRS, European isolates, North American isolates, real-time RT-PCR, serum, quantification, porcine GAPDH, cyclophilin, housekeeping genes


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