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Development and validation of an ovine progressive pneumonia virus quantitative PCR.

ABSTRACT: Ovine progressive pneumonia virus (OPPV) infects at least one sheep in 81% of U.S. sheep flocks, as determined by serology, and can cause viral mastitis, arthritis, dyspnea, and cachexia. Diagnostic tests that quantify OPPV proviral load in peripheral blood leukocytes (PBL) provide an additional method for identification of infected sheep and may help to further understanding of the pathogenesis of OPPV-induced disease. In this study, we compared a new OPPV real-time quantitative PCR (qPCR) assay specific for the transmembrane region of the envelope gene (tm) with a competitive inhibition enzyme-linked immunosorbent assay (cELISA) using 396 PBL samples and sera from Idaho sheep. The OPPV qPCR had a positive concordance of 96.2% +/- 2.3% and a negative concordance of 97.7% +/- 2.5% compared to the cELISA, with a kappa value of 0.93, indicating excellent agreement between the two tests. In addition, the presence of tm in the three OPPV qPCR-positive and cELISA-negative sheep and in 15 sheep with different OPPV proviral loads was confirmed by cloning and sequencing. These data indicate that the OPPV qPCR may be used as a supplemental diagnostic tool for OPPV infection and for measurement of viral load in PBLs of infected sheep.

SUBMITTER: Herrmann-Hoesing LM

PROVIDER: S-EPMC2168119 | biostudies-literature | 2007 Oct

REPOSITORIES: biostudies-literature

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Development and validation of an ovine progressive pneumonia virus quantitative PCR.

Herrmann-Hoesing Lynn M LM White Stephen N SN Lewis Gregory S GS Mousel Michelle R MR Knowles Donald P DP

Clinical and vaccine immunology : CVI 20070815 10

Ovine progressive pneumonia virus (OPPV) infects at least one sheep in 81% of U.S. sheep flocks, as determined by serology, and can cause viral mastitis, arthritis, dyspnea, and cachexia. Diagnostic tests that quantify OPPV proviral load in peripheral blood leukocytes (PBL) provide an additional method for identification of infected sheep and may help to further understanding of the pathogenesis of OPPV-induced disease. In this study, we compared a new OPPV real-time quantitative PCR (qPCR) assa ...[more]

PMID: 17699832

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Project description:Nicotiana benthamiana is the most widely-used experimental host in plant virology. The recent release of the draft genome sequence for N. benthamiana consolidates its role as a model for plant-pathogen interactions. Quantitative real-time PCR (qPCR) is commonly employed for quantitative gene expression analysis. For valid qPCR analysis, accurate normalisation of gene expression against an appropriate internal control is required. Yet there has been little systematic investigation of reference gene stability in N. benthamiana under conditions of viral infections. In this study, the expression profiles of 16 commonly used housekeeping genes (GAPDH, 18S, EF1?, SAMD, L23, UK, PP2A, APR, UBI3, SAND, ACT, TUB, GBP, F-BOX, PPR and TIP41) were determined in N. benthamiana and those with acceptable expression levels were further selected for transcript stability analysis by qPCR of complementary DNA prepared from N. benthamiana leaf tissue infected with one of five RNA plant viruses (Tobacco necrosis virus A, Beet black scorch virus, Beet necrotic yellow vein virus, Barley stripe mosaic virus and Potato virus X). Gene stability was analysed in parallel by three commonly-used dedicated algorithms: geNorm, NormFinder and BestKeeper. Statistical analysis revealed that the PP2A, F-BOX and L23 genes were the most stable overall, and that the combination of these three genes was sufficient for accurate normalisation. In addition, the suitability of PP2A, F-BOX and L23 as reference genes was illustrated by expression-level analysis of AGO2 and RdR6 in virus-infected N. benthamiana leaves. This is the first study to systematically examine and evaluate the stability of different reference genes in N. benthamiana. Our results not only provide researchers studying these viruses a shortlist of potential housekeeping genes to use as normalisers for qPCR experiments, but should also guide the selection of appropriate reference genes for gene expression studies of N. benthamiana under other biotic and abiotic stress conditions.

Dataset Information

Development and validation of an ovine progressive pneumonia virus quantitative PCR.

Publications

Development and validation of an ovine progressive pneumonia virus quantitative PCR.

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