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Comparison of a LightCycler-based real-time PCR for quantitation of Epstein-Barr viral load in different clinical specimens with semiquantitative PCR

2006; Wiley; Volume: 78; Issue: 5 Linguagem: Inglês

10.1002/jmv.20581

1096-9071

Andi Krumbholz, Astrid Meerbach, Roland Zell, Bernd Gruhn, Andreas Henke, Eckhard Birch‐Hirschfeld, P Wutzler,

Parvovirus B19 Infection Studies

Journal of Medical VirologyVolume 78, Issue 5 p. 598-607 Research Article Comparison of a LightCycler-based real-time PCR for quantitation of Epstein-Barr viral load in different clinical specimens with semiquantitative PCR Andi Krumbholz, Corresponding Author Andi Krumbholz [email protected] Institute of Virology and Antiviral Therapy, Medical Center, Friedrich Schiller University Jena, Jena, GermanyInstitute of Virology and Antiviral Therapy, Medical Center, Friedrich Schiller University Jena, Hans Knöll Strasse 2, D-07745 Jena, Germany.===Search for more papers by this authorAstrid Meerbach, Astrid Meerbach Institute of Virology and Antiviral Therapy, Medical Center, Friedrich Schiller University Jena, Jena, GermanySearch for more papers by this authorRoland Zell, Roland Zell Institute of Virology and Antiviral Therapy, Medical Center, Friedrich Schiller University Jena, Jena, GermanySearch for more papers by this authorBernd Gruhn, Bernd Gruhn Department of Pediatrics, Medical Center, Friedrich Schiller University Jena, Jena, GermanySearch for more papers by this authorAndreas Henke, Andreas Henke Institute of Virology and Antiviral Therapy, Medical Center, Friedrich Schiller University Jena, Jena, GermanySearch for more papers by this authorEckhard Birch-Hirschfeld, Eckhard Birch-Hirschfeld Institute of Virology and Antiviral Therapy, Medical Center, Friedrich Schiller University Jena, Jena, GermanySearch for more papers by this authorPeter Wutzler, Peter Wutzler Institute of Virology and Antiviral Therapy, Medical Center, Friedrich Schiller University Jena, Jena, GermanySearch for more papers by this author Andi Krumbholz, Corresponding Author Andi Krumbholz [email protected] Institute of Virology and Antiviral Therapy, Medical Center, Friedrich Schiller University Jena, Jena, GermanyInstitute of Virology and Antiviral Therapy, Medical Center, Friedrich Schiller University Jena, Hans Knöll Strasse 2, D-07745 Jena, Germany.===Search for more papers by this authorAstrid Meerbach, Astrid Meerbach Institute of Virology and Antiviral Therapy, Medical Center, Friedrich Schiller University Jena, Jena, GermanySearch for more papers by this authorRoland Zell, Roland Zell Institute of Virology and Antiviral Therapy, Medical Center, Friedrich Schiller University Jena, Jena, GermanySearch for more papers by this authorBernd Gruhn, Bernd Gruhn Department of Pediatrics, Medical Center, Friedrich Schiller University Jena, Jena, GermanySearch for more papers by this authorAndreas Henke, Andreas Henke Institute of Virology and Antiviral Therapy, Medical Center, Friedrich Schiller University Jena, Jena, GermanySearch for more papers by this authorEckhard Birch-Hirschfeld, Eckhard Birch-Hirschfeld Institute of Virology and Antiviral Therapy, Medical Center, Friedrich Schiller University Jena, Jena, GermanySearch for more papers by this authorPeter Wutzler, Peter Wutzler Institute of Virology and Antiviral Therapy, Medical Center, Friedrich Schiller University Jena, Jena, GermanySearch for more papers by this author First published: 22 March 2006 https://doi.org/10.1002/jmv.20581Citations: 8AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Abstract Measurement of viral load is important in predicting and monitoring of Epstein-Barr virus (EBV)-associated diseases especially in immunocompromised patients. The objectives of this study were the development of a LightCycler-based real-time PCR assay using primers and probes which recognize the virus capsid antigen p23-encoding region and its comparison to the semiquantitative PCR. The LightCycler protocol shows a high degree of specificity and inter- and intra-assay reproducibility. Concerning sensitivity, a good correlation between both methods was demonstrated for standard plasmid DNA, reference DNA isolated from the EBV-genome containing Namalwa cell line, and DNA extracted from plasma/cerebrospinal fluid (CSF). The detection limit was determined with 1 copy/µl eluate for the standard plasmid DNA and with 500 copies/ml plasma or CSF. For DNA derived from peripheral blood mononuclear cells (PBMCs), a decrease of sensitivity by factor 10–100 was found when larger amounts of background DNA (500 and 100 ng) were used presuming an inhibitory effect of cellular DNA. This was supported by running dilutions of the plasmid standard carried out with EBV-negative Ramos cell DNA. Thus, the cut-off level was estimated with 100–500 copies/105 PBMCs, when 50 or 10 ng total DNA were tested. The results indicate that the real-time PCR described here is a first line tool for the determination of viral load in plasma and CSF. Semiquantitative nested PCR is used for screening of PBMCs viral load. Positive specimens containing more than 500 copies/105 cells are measured for exact values by real-time PCR. To circumvent inhibitory effects of cellular DNA, measurements should be carried out generally with 50–10 ng DNA. J. Med. Virol. 78:598–607, 2006. © 2006 Wiley-Liss, Inc. 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