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

Meav-Lang J Lay1*, Robyn M Lucas2, Mala Ratnamohan1, Janette Taylor1, Anne-Louise Ponsonby3, Dominic E Dwyer1 and the Ausimmune Investigator Group (AIG)

Author affiliations

1 Virology Department, Centre For Infectious Diseases & Microbiology Laboratory Services, Institute of Clinical Pathology & Medical Research, Institute Road, Westmead Hospital, Westmead 2145, New South Wales, Australia

2 National Centre for Epidemiology and Population Health, The Australian National University, Canberra, ACT, 0200 Australia

3 Murdoch Childrens Research Institute, 9th Floor AP Building, The Royal Children's Hospital, Flemington Road, Parkville, Victoria 3052, Australia

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Citation and License

Virology Journal 2010, 7:252  doi:10.1186/1743-422X-7-252

Published: 22 September 2010

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 × 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).

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.