A multiplex calibrated real-time PCR assay for quantitation of DNA of EBV-1 and 2

Francesca Gatto, Giulia Cassina, Francesco Broccolo, Giuseppe Morreale, Edoardo Lanino, Eddi Di Marco, Efthiya Vardas, Daniela Bernasconi, Stefano Buttò, Nicola Principi, Susanna Esposito, Gabriella Scarlatti, Paolo Lusso, Mauro S. Malnati

Research output: Contribution to journalArticle

Abstract

Accurate and highly sensitive tests for the diagnosis of active Epstein-Barr virus (EBV) infection are essential for the clinical management of individuals infected with EBV. A calibrated quantitative real-time PCR assay for the measurement of EBV DNA of both EBV-1 and 2 subtypes was developed, combining the detection of the EBV DNA and a synthetic DNA calibrator in a multiplex PCR format. The assay displays a wide dynamic range and a high degree of accuracy even in the presence of 1 μg of human genomic DNA. This assay measures with the same efficiency EBV DNA from strains prevalent in different geographic areas. The clinical sensitivity and specificity of the system were evaluated by testing 181 peripheral blood mononuclear cell (PBMCs) and plasma specimens obtained from 21 patients subjected to bone marrow transplantation, 70 HIV-seropositive subjects and 23 healthy controls. Patients affected by EBV-associated post-transplant lymphoprolipherative disorders had the highest frequency of EBV detection and the highest viral load. Persons infected with HIV had higher levels of EBV DNA load in PBMCs and a higher frequency of EBV plasma viremia compared to healthy controls. In conclusion, this new assay provides a reliable high-throughput method for the quantitation of EBV DNA in clinical samples.

Original languageEnglish
Pages (from-to)98-105
Number of pages8
JournalJournal of Virological Methods
Volume178
Issue number1-2
DOIs
Publication statusPublished - Dec 2011

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Keywords

  • EBV DNA load
  • HIV-1
  • Multiplex PCR
  • Real-time PCR

ASJC Scopus subject areas

  • Virology

Cite this

Gatto, F., Cassina, G., Broccolo, F., Morreale, G., Lanino, E., Di Marco, E., Vardas, E., Bernasconi, D., Buttò, S., Principi, N., Esposito, S., Scarlatti, G., Lusso, P., & Malnati, M. S. (2011). A multiplex calibrated real-time PCR assay for quantitation of DNA of EBV-1 and 2. Journal of Virological Methods, 178(1-2), 98-105. https://doi.org/10.1016/j.jviromet.2011.08.022