Measurement of Epstein-Barr virus DNA load using a novel quantification standard containing two EBV DNA targets and SYBR Green I dye

Abstract

BackgroundReactivation of Epstein-Barr virus (EBV) infection may cause serious, life-threatening complications in immunocompromised individuals. EBV DNA is often detected in EBV-associated disease states, with viral load believed to be a reflection of virus activity. Two separate real-time quantitative polymerase chain reaction (QPCR) assays using SYBR Green I dye and a single quantification standard containing two EBV genes, Epstein-Barr nuclear antigen-1 (EBNA-1) and BamHI fragment H rightward open reading frame-1 (BHRF-1), were developed to detect and measure absolute EBV DNA load in patients with various EBV-associated diseases. EBV DNA loads and viral capsid antigen (VCA) IgG antibody titres were also quantified on a population sample.ResultsEBV DNA was measurable in ethylenediaminetetraacetic acid (EDTA) whole blood, peripheral blood mononuclear cells (PBMCs), plasma and cerebrospinal fluid (CSF) samples. EBV DNA loads were detectable from 8.0 × 102 to 1.3 × 108 copies/ml in post-transplant lymphoproliferative disease (n = 5), 1.5 × 103 to 2.0 × 105 copies/ml in infectious mononucleosis (n = 7), 7.5 × 104 to 1.1 × 105 copies/ml in EBV-associated haemophagocytic syndrome (n = 1), 2.0 × 102 to 5.6 × 103 copies/ml in HIV-infected patients (n = 12), and 2.0 × 102 to 9.1 × 104 copies/ml in the population sample (n = 218). EBNA-1 and BHRF-1 DNA were detected in 11.0% and 21.6% of the population sample respectively. There was a modest correlation between VCA IgG antibody titre and BHRF-1 DNA load (rho = 0.13, p = 0.05) but not EBNA-1 DNA load (rho = 0.11, p = 0.11).ConclusionTwo sensitive and specific real-time PCR assays using SYBR Green I dye and a single quantification standard containing two EBV DNA targets, were developed for the detection and measurement of EBV DNA load in a variety of clinical samples. These assays have application in the investigation of EBV-related illnesses in immunocompromised individuals.

Epstein-Barr Virus Genotypes and Strains in Central Nervous System Demyelinating Disease and Epstein-Barr Virus-Related Illnesses in Australia

“…The real-time PCR assay could detect samples with EBV DNA loads as low as 2.0 × 10 2 copies/ml as previously shown [18]. The generation of single, distinct bands upon electrophoretic separation confirmed primer specificity and infection with a single genotype.…”

“…Five individuals in group 3 had multiple samples: three plasma samples were available for 1 IM patient while 2–6 different specimen types (EDTA, plasma and CSF) were accessible for 4 LPD patients. Genotyping studies were conducted retrospectively following EBV DNA load and serological testing [18]; all samples were from individuals with detectable EBV viral capsid antigen immunoglobulin G (VCA IgG) and were quantitative-PCR positive for at least one EBV DNA target (EBNA-1 or BHRF-1). …”

Changes in Tonsil B Cell Phenotypes and EBV Receptor Expression in Children Under 5‐Years‐Old

“…The EBV viral load was much higher per cell (3.2 3 10 6 copies/ 10 6 TMC) than has been reported in peripheral blood mononuclear cells (44,45). The EBV viral load was much higher per cell (3.2 3 10 6 copies/ 10 6 TMC) than has been reported in peripheral blood mononuclear cells (44,45).…”

“…We observed a relatively high frequency of EBV infection during the first five years of life (60%). The EBV viral load was much higher per cell (3.2 3 10 6 copies/ 10 6 TMC) than has been reported in peripheral blood mononuclear cells (44,45). This observation is not surprising considering that tonsils are the site of EBV entry and persistence in the lymphocyte compartment (46).…”

Resveratrol Prevents EBV Transformation and Inhibits the Outgrowth of EBV-Immortalized Human B Cells

“…Amplification of cDNA was monitored using the SYBR Premix Ex Taq RT-PCR kit (Catalog RR041, Takara Bio, Japan) on a StepOne plus instrument (Applied Biosystems). Specific primers for BHRF1 and EBNA-1 [20], and LMP1 [21], as well as a GAPDH primer set (Takara Bio) were used for mRNA quantification in each sample. The amount of each mRNA relative to GAPDH mRNA was calculated by the comparative CT method using the relative expression function included in the StepOne v2.2 software package (Applies Biosystems).…”