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Journal of Clinical Microbiology, November 2002, p. 3986-3992, Vol. 40, No. 11
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.11.3986-3992.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Comparison of Quantitative Competitive PCR with LightCycler-Based PCR for Measuring Epstein-Barr Virus DNA Load in Clinical Specimens

Servi J. C. Stevens, Sandra A. W. M. Verkuijlen, Adriaan J. C. van den Brule, and Jaap M. Middeldorp* Received 11 February 2002/ Returned for modification 25 May 2002/ Accepted 17 August 2002

The aim of this study was to develop a LightCycler-based real-time PCR assay for monitoring the Epstein-Barr virus (EBV) DNA load in unfractionated whole blood. This assay was compared with quantitative competitive PCR (Q-PCR) for EBV. The LightCycler-based assay was highly sensitive and reproducible when quantifying plasmid DNA in either the presence or absence of healthy donor blood DNA. Amplifying plasmid DNA in DNA backgrounds from different donors slightly increased the variation of quantification, indicating that clinical specimen DNA has an influence on quantification. In most transplant recipients, a good correlation was observed between EBV DNA load dynamics determined by LightCycler and Q-PCR in follow-up samples, although the correlation between absolute values of EBV DNA loads was weak and occasional samples were false negative in the LightCycler assay. In 253 cross-sectional blood samples from patients with Burkitt's lymphoma, infectious mononucleosis, or human immunodeficiency virus infection, a weak but significant correlation between the two methods was found (r2 = 0.37, P < 0.001). Our results indicate that the clinical specimen DNA background may influence the absolute values of EBV DNA load in LightCycler analyses but that this effect is rare. LightCycler PCR is very well suited for monitoring of EBV DNA load dynamics, and its diagnostic value is comparable to that of Q-PCR. To avoid false negativity or underestimation of viral load, future internal calibration of the LightCycler is recommended. This would also enhance EBV load assay standardization and interinstitute comparisons.

* Corresponding author. Mailing address: Department of Pathology, University Hospital Vrije Universiteit, De Boelelaan 1117, 1081 HV Amsterdam, The Netherlands. Phone: 31 20 4444001. Fax: 31 20 4442964. E-mail: j.middeldorp{at}vumc.nl.

Journal of Clinical Microbiology, November 2002, p. 3986-3992, Vol. 40, No. 11
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.11.3986-3992.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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