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Journal of Clinical Microbiology, February 2001, p. 564-569, Vol. 39, No. 2
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.2.564-569.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Normalized Quantification by Real-Time PCR of Epstein-Barr Virus Load in Patients at Risk for Posttransplant Lymphoproliferative Disorders

Wolfram J. Jabs,1,* Holger Hennig,1 Michael Kittel,2 Klaus Pethig,3 Françoise Smets,4 Peter Bucsky,2 Holger Kirchner,1 and Hans J. Wagner2,dagger

Institute of Immunology and Transfusion Medicine1 and Department of Pediatrics,2 University of Lübeck School of Medicine, Lübeck, and Department of Thoracic Surgery, Hannover School of Medicine, Hannover,3 Germany, and Department of Pediatrics, Université Catholique de Louvain, Brussels, Belgium4

Received 2 August 2000/Returned for modification 16 October 2000/Accepted 30 November 2000

The load of Epstein-Barr virus (EBV) in peripheral blood mononuclear cells of transplant recipients represents a predictive parameter for posttransplant lymphoproliferative disorders (PTLD). The aim of our work was to develop a rapid and reliable PCR protocol for the quantification of cell-associated EBV DNA in transplant recipients. In contrast to previous studies, a protocol that facilitated quantification independent of photometric nucleic acid analysis was established. We took advantage of the real-time PCR technology which allows for single-tube coamplification of EBV and genomic C-reactive protein (CRP) DNA. EBV copy numbers were normalized by division by the amount of CRP DNA, with the quotient representing the actual amount of amplifiable genomic DNA per reaction. Coamplification of CRP DNA did not result in a diminished detection limit for EBV. By using the protocol without normalization, EBV copy numbers in 4 out of 10 PTLD patients were within the normal range determined with data for 114 transplant recipients that served as controls. After normalization, however, all of the PTLD patients had a higher viral load than the control population, indicating an increased sensitivity of the assay. Moreover, EBV copy numbers obtained for one patient by conventional quantification and suggestive of relapsing PTLD were within normal range after normalization. We conclude that normalization of PCR signals to coamplified genomic DNA allows a more accurate quantification of cell-bound EBV.

* Corresponding author. Present address: 1st Department of Internal Medicine, University of Lübeck School of Medicine, Ratzeburger Allee 160, 23538 Lübeck, Germany. Phone: 49-451-500 2359. Fax: 49-451-500 3402. E-mail: jabs{at}immu.mu-luebeck.de.

dagger Present address: Department of Microbiology and Immunology, Louisiana State University Health Science Center, Shreveport, Louisiana.

Journal of Clinical Microbiology, February 2001, p. 564-569, Vol. 39, No. 2
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.2.564-569.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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