Establishment of Real-Time Polymerase Chain Reaction-Based Assay for Quantitation of Epstein-Barr Virus DNA in Healthy Donors and in Patients with EBV Associated Lymphoma
Patcha Incomserb MSc*, Parvapan Bhattarakosol PhD**, Wanla Kulwichit MD***, Wasun Chantratita PhD****, Pokrath Hansasuta MD, DPhil (Oxon)**
Affiliation : * Inter-department of Medical Microbiology, Faculty of Graduate School, Chulalongkorn University ** Department of Microbiology, Faculty of Medicine, Chulalongkorn University *** Department of Internal Medicine, Faculty of Medicine, Chulalongkorn University **** Department of Pathology, Faculty of Medicine,Mahidol University
Epstein-Barr virus (EBV) is associated with several malignancies including nasopharyngeal carci- noma and lymphoma in immunocompromised patients. Quantitative monitoring of EBV DNA in these patients has recently become essential for management of the disease. In the present study the authors developed a rapid and reliable real-time PCR to quantify the EBV DNA in peripheral blood mononuclear cell (PBMC) using hybridization probe technique. The real-time primers and probes in this real-time PCR system were designed based on EBNA-1 sequence. The newly-established real-time PCR demonstrated its high sensitivity (as few as 10 copies of EBV could be detected) and specificity. The intra- and inter-assay variations of the assay were shown to be within a 0.5-log10-difference range. A total of 2 EBV-seronegative, 14 EBV-seropositive healthy donors and 4 patients with PCNSL were enrolled into the study. Our results revealed the median of EBV-DNA in lymphoma patients (7,886 copies/106 PBMC or 15,150 copies /µg DNA) was higher than that of healthy donors (<10 copies/106 PBMC or <10 copies/µg DNA) with statistic significance (P<0.01). Assess- ment of this assay in larger number of donors and patients will provide clinical cut-off values which are essential for monitoring and diagnosis of EBV-associated diseases.
Keywords : EBV-DNA, Real-time PCR
