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

Abstract

BACKGROUND:

Reactivation 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.

RESULTS:

EBV 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 × 10(2) to 1.3 × 10(8) copies/ml in post-transplant lymphoproliferative disease (n = 5), 1.5 × 10(3) to 2.0 × 10(5) copies/ml in infectious mononucleosis (n = 7), 7.5 × 10(4) to 1.1 × 10(5) copies/ml in EBV-associated haemophagocytic syndrome (n = 1), 2.0 × 10(2) to 5.6 × 10(3) copies/ml in HIV-infected patients (n = 12), and 2.0 × 10(2) to 9.1 × 10(4) 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).

CONCLUSION:

Two 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.

• Authors: Meav-Lang J Lay, Robyn M Lucas, Mala Ratnamohan, Janette Taylor, Anne-Louise Ponsonby, Dominic E Dwyer, Ausimmune Investigator Group (AIG)
• Journal: Virology journal (Virol J) Vol. 7 Pg. 252 (Sep 22 2010) ISSN: 1743-422X [Electronic] England
• PMID: 20860842 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Antibodies, Viral
• Benzothiazoles
• Diamines
• Immunoglobulin G
• Organic Chemicals
• Quinolines
• SYBR Green I

Topics

• Adolescent
• Adult
• Antibodies, Viral (blood)
• Benzothiazoles
• Blood (virology)
• Cerebrospinal Fluid (virology)
• Diamines
• Epstein-Barr Virus Infections (virology)
• Female
• Herpesvirus 4, Human (isolation & purification)
• Humans
• Immunoglobulin G (blood)
• Leukocytes, Mononuclear (virology)
• Male
• Middle Aged
• Organic Chemicals (metabolism)
• Plasma (virology)
• Quinolines
• Reference Standards
• Sensitivity and Specificity
• Staining and Labeling (methods)
• Viral Load (methods, standards)
• Young Adult