Abstract
BackgroundQuantification 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.MethodsPlasmids 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.ResultsThe assay was specific and showed satisfactory efficiency (82.2%, R2=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 (CT) < 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 CT (mean ± SD) of 33.8 ± 1.77 in healthy individuals and 2.06 – 8.49 log copies/mL of plasma with CT (mean ± SD) of 24.3 ± 1.04 in HSCT recipients.ConclusionSYBR 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.
Highlights
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 report the validation of SYBR Green based quantification assay for routine use by using a set of primer pairs targeted for amplifying a well-conserved sequence of ORF-2 [14]
The observed melting curve (Melting temperatureTM: 85.01°C ± 0.27 that was clearly different from the melting curve of primer-dimers (TM ranging from 71.1 – 72.9°C) as well as the purity of the TTV-specific 96 bp amplicon in the samples that contained TTV DNA (Figure 3) confirmed the high specificity of the PCR
Summary
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. Viral titers in the plasma may reflect the individual’s immune status, since immunocompromised patients harbor high loads of TTV [4]. This approach can be used to estimate immune recovery in TTV DNA has a total genomic length of approximately 3.8 kilobases [6,7,8,9] and contains two large-open reading frames (ORF1 and ORF2) and several smaller ORFs [3]. The conserved ORF-2 region allows the design of primers expected to amplify most strains of TTV [14]
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