VALIDATION OF ORAGENE DNA SELF- COLLECTION KIT FOR REAL-TIME EBV DNA VIRAL LOAD

Background:  Quantification of Epstein Barr virus (EBV) is important for the diagnosis and management of serious EBV diseases.  A variety of sample types are utilized for quantifying EBV DNA levels including peripheral blood, serum, plasma and CSF; all require invasive collection procedures.  We describe a non-invasive collection of human saliva using the commercially available Oragene DNA Self-Collection Kits for real-time monitoring of EBV DNA viral load.

Methods:  Saliva samples were collected from 11 healthy subjects and spiked with ~10,000 c/ml  00 infectious EBV.  Oral supernatant and buccal epithelial cells were separated by centrifugation, aliquoted into Oragene kit (DNA Genotek Inc) and shipped room temp to ViraCor testing facility.  0.2 ml sample was extracted using Qiagen DNA blood mini kit according to manufacturer’s instructions.  For each sample purified DNA was eluted in 50 ul; 10 ul was amplified in duplicate using the ABI 7500 real-time PCR platform for quantification (QPCR) of EBV genomic DNA.  The number of EBV c/ml was determined by extrapolation of C_T values from a standard curve constructed using cloned plasmid DNA for the target analyte EBNA-1.  Stability testing was performed on samples serially collected from stored specimens over a period of 10 days to 3 weeks at 2 – 8°C.

Results:  QPCR assay results demonstrated robust proficiency with intra-subject standard deviation (SD) of 0.02 log c/ml for oral supernatant and 0.06 log c/mL for oral cell fractions.  One subject’s oral supernatant prior to EBV spike   had much higher EBV DNA levels compared to other subjects and was excluded from the analyses of intra-subject variability.  Mean oral supernatant EBV DNA for all subjects excluding outlier (n=10) was 4.31 log/ml, + 0.181 SD and a coefficient of variation (CV) of 4.2%.  Mean cell EBV DNA levels were 3.36 log/ml, + 0.305 SD with a CV of 9.06%.  Stability testing on a subset of samples, n=6, revealed SD ranging from 0.04 to 0.10 log with %CV of  0.75% to 2.81% for oral supernatants and 0.05 to 0.26 log SD with %CV of 0.76% to 8.09% for cell fractions.  

Conclusion:  Oragene DNA Self-Collection kit demonstrated excellent recovery of EBV DNA from human saliva for the downstream application of real-time DNA QPCR and is well suited for saliva self-collection and room temp shipping.  The ViraCor real-time EBV DNA viral load assay demonstrated robust proficiency and excellent reproducibility in determining EBV DNA levels from human saliva.