Validation of SYBR Green based quantification assay for the detection of human Torque Teno virus titers from plasma

Background: Quantification of titers of ubiquitous viruses such as Torque teno virus (TTV) that do not cause clinical symptoms might be helpful in assessing the immune status of an individual. We hereby describe the validation of a SYBR Green-based TTV quantification method for plasma samples. Methods: Plasmids with TTV specific inserts were used for preparing standards and absolute quantification of TTV was performed using SYBR Green methodology. The method was assessed for its accuracy and precision (intra and inter-day) on four non-consecutive days. TTV was also quantified from plasma samples of 20 healthy volunteers and from 30 hematopoietic stem cell transplant (HSCT) recipients. Results: The assay was specific and showed satisfactory efficiency (82.2%, R-2=0.99) with the limit of quantification defined as 100 copies per reaction. The assay had good precision (inter and intra-day coefficient of variation in cycle threshold (C-T) < 4%) and accuracy (100 +/- 10%) in the range of 100 to 1010 copies/reaction. We found TTV loads ranging from 2.5 - 4.07 log copies/mL of plasma with C-T (mean +/- SD) of 33.8 +/- 1.77 in healthy individuals and 2.06 - 8.49 log copies/mL of plasma with C-T (mean +/- SD) of 24.3 +/- 1.04 in HSCT recipients. Conclusion: SYBR Green-based q-PCR assay combines simplicity with satisfactory sensitivity and may be suitable for monitoring the immune status of transplant recipients, where TTV loads over time may serve as a marker for immune reconstitution in human plasma samples.