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Quantitation of Epstein–Barr virus mRNA using reverse transcription and real‐time PCR
Author(s) -
Weinberger Birgit,
Plentz Annelie,
Weinberger Klaus M.,
Hahn Joachim,
Holler Ernst,
Jilg Wolfgang
Publication year - 2004
Publication title -
journal of medical virology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.782
H-Index - 121
eISSN - 1096-9071
pISSN - 0146-6615
DOI - 10.1002/jmv.20220
Subject(s) - lytic cycle , virology , biology , virus , reverse transcriptase , rna , microbiology and biotechnology , epstein–barr virus , taqman , real time polymerase chain reaction , dna , gene , biochemistry , genetics
Monitoring of Epstein–Barr virus (EBV) infection and reactivation in immunocompromized patients (e.g., after organ or bone‐marrow transplantation) is based mainly on serological assays and detection of viral DNA. For further characterization of virus reactivation and monitoring of viral transcription we established real‐time RT‐PCR assays using TaqMan technology to sensitively quantify viral transcripts expressed at different times of the lytic cycle: for BZLF1, an immediate early transactivator initiating the transition from latency to lytic replication, for the DNA‐polymerase BALF5 and for the major viral glycoprotein gp350/220 (BLLF1). RNA‐isolation was optimized to eliminate contaminating DNA. Preparations were shown to be virtually DNA‐free for up to 10 6 copies of RNA. With our PCR systems, it is possible to detect 10 copies of DNA or 100 copies of RNA per reaction as shown with serial dilutions of DNA‐plasmids or in vitro transcribed RNA, respectively. This corresponds to a detection limit of 8 × 10 2 copies/10 6 peripheral blood mononuclear cells (PBMCs). Evaluation of this system showed that even in healthy carriers borderline levels of BLLF1 mRNA were sometimes detectable. In patients with acute infectious mononucleosis (IM) viral transcripts were regularly found in varying concentrations. Extremely high levels of all three mRNA species could be seen in a patient after bone‐marrow transplantation monitored during an episode of lymphoproliferation which regressed during treatment with acyclovir and transfusion of donor T‐cells. This sensitive and reproducible method to detect and quantify different transcripts of EBV can be used to closely monitor reactivation of EBV, e.g., in immunocompromized patients. J. Med. Virol. 74:612–618, 2004. © 2004 Wiley‐Liss, Inc.