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


CONCLUSIONS
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.
Lay
Meav-Lang J
ML
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. mlay4697@uni.sydney.edu.au
Lucas
Robyn M
RM
Ratnamohan
Mala
M
Taylor
Janette
J
Ponsonby
Anne-Louise
AL
Dwyer
Dominic E
DE
D000293:Adolescent; D000328:Adult; D000914:Antibodies, Viral / Q000097:blood; D052160:Benzothiazoles; D001769:Blood / Q000821:virology; D002555:Cerebrospinal Fluid / Q000821:virology; D003959:Diamines; D020031:Epstein-Barr Virus Infections / Q000821:virology*; D005260:Female; D004854:Herpesvirus 4, Human / Q000302:isolation & purification*; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D007963:Leukocytes, Mononuclear / Q000821:virology; D008297:Male; D008875:Middle Aged; D009930:Organic Chemicals / Q000378:metabolism*; D010949:Plasma / Q000821:virology; D011804:Quinolines; D012015:Reference Standards; D012680:Sensitivity and Specificity; D013194:Staining and Labeling / Q000379:methods; D019562:Viral Load / Q000379:methods* / Q000592:standards*; D055815:Young Adult
D000914:Antibodies, Viral; D052160:Benzothiazoles; D003959:Diamines; D007074:Immunoglobulin G; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I