Validation of SYBR Green based quantification assay for the detection of human Torque Teno virus titers from plasma
© Tyagi et al.; licensee BioMed Central Ltd. 2013
Received: 19 April 2013
Accepted: 7 June 2013
Published: 11 June 2013
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.
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.
The 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.
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.
Torque teno virus (TTV), classified into the family anelloviridae, genus alphatorquevirus, was first described in a patient with non-A-E hepatitis [1, 2]. TTV is a non-enveloped, single-stranded, circular DNA virus present in plasma of >90% of individuals, regardless of geographical origin, age or health status [2, 3]. Viral titers in the plasma may reflect the individual’s immune status, since immunocompromised patients harbor high loads of TTV . This approach can be used to estimate immune recovery in hematopoietic stem cell transplant (HSCT) recipients by monitoring TTV titers after transplantation .
TTV DNA has a total genomic length of approximately 3.8 kilobases [6–9] and contains two large-open reading frames (ORF1 and ORF2) and several smaller ORFs . TTV exhibits a wide range of sequence variability, with at least 38 TTV genotypes and forms five distinct phylogenetic groups [10–13]. The conserved ORF-2 region allows the design of primers expected to amplify most strains of TTV .
Several previously described quantification methods for human TTV [15–17] are based on TaqMan technology, which may be less suitable for quantification of highly variable viruses such as TTV. SYBR Green-based PCR with primers annealing to more conserved regions may be preferable. In the present study, 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 .
Results and discussion
SYBR Green real-time PCR assay for TTV using two independent standards series
Plasmid – standards
CTMean ± SD (n=2)*
10 × 109
4.11 ± 0.09
4.17 ± 0.08
4.11 ± 0.09
4.09 ± 0.02
10 × 108
7.14 ± 0.07
7.24 ± 0.22
7.08 ± 0.06
7.11 ± 0.10
10 × 107
11.15 ± 0.05
11.03 ± 0.16
11.26 ± 0.12
11.19 ± 0.06
10 × 106
15.21 ± 0.03
15.10 ± 0.02
15.19 ± 0.01
15.19 ± 0.003
10 × 105
19.15 ± 0.009
19.03 ± 0.06
19.14 ± 0.02
19.11 ± 0.02
10 × 104
22.89 ± 0.009
22.86 ± 0.09
22.98 ± 0.13
23.00 ± 0.05
10 × 103
26.95 ± 0.06
26.87 ± 0.04
26.95 ± 0.005
27.02 ± 0.13
10 × 102
30.83 ± 0.16
30.89 ± 0.08
31.08 ± 0.03
30.97 ± 0.13
10 × 101
34.29 ± 0.30
33.78 ± 0.57
33.93 ± 0.77
33.87 ± 0.56
SYBR Green PCR assay for TTV using single series of plasmid standards
CTMean ± SD (n=3)*
10 × 109
4.21 ± 0.02
4.18 ± 0.01
10 × 108
7.59 ± 0.05
7.52 ± 0.05
10 × 107
11.74 ± 0.05
11.56 ± 0.01
10 × 106
15.83 ± 0.04
15.56 ± 0.01
10 × 105
19.75 ± 0.05
19.37 ± 0.01
10 × 104
24.15 ± 0.03
23.64 ± 0.02
10 × 103
28.40 ± 0.03
27.75 ± 0.05
10 × 102
33.21 ± 0.20
32.51 ± 0.06
10 × 101
35.59 ± 0.39
35.25 ± 0.06
Several TTV studies using TaqMan chemistry have reported varying levels of sensitivity, ranging from 120 to 1000 copies/mL for different types of clinical specimens [5, 14, 15, 17–22], which may be the result of differences in the primers used. Although sequence heterogeneity in TTV is high with some variants only sharing 50% of nucleotides [23, 24], certain conserved regions can be chosen for primer design in order to amplify more than one subtype of TTV . Focosi et al.  and Maggi et al.  used probes directed against the conserved portion of untranslated region (UTR), while others used probes specific for highly conserved region of ORF2 and ORF1 of TTV [14, 15, 17].
This report describes the validation of a SYBR Green assay for quantification of TTV viral load in human plasma samples. The developed assay was accurate with satisfactory efficiency, reproducibility in the range of 100-1010 copies/ reaction. This simple assay can be used in studies assessing TTV plasma loads as a marker of immune reconstitution. A prospective study is currently ongoing in our institution to validate the correlation of TTV titer and the immune status post HSCT.
Reagents and consumables
Oligonucleotide primers used for human torque teno virus (TTV)
Sequence (5` to 3`)
Moen EM et al.
Samples and DNA extraction
Blood samples (5 mL) collected in EDTA tubes from 20 healthy adult volunteers and 30 randomly selected adult HSCT recipients were centrifuged at 900 g for 10 minutes to separate plasma which was immediately frozen at -20°C until used for DNA extraction. Two independent DNA extractions were performed for each of the healthy individuals along with one independent DNA extraction for HSCT recipients, each from 200 μl of plasma using QIAamp MinElute Virus Spin kit according to the manufacturer’s recommendations. DNA was eluted in 30 μL of Milli-Q water. All extracted DNA samples were stored at -20°C until the analysis. The study protocol was approved by the institution’s ethics committee and healthy donors and HSCT recipient’s samples were used after obtaining informed consent.
Construction of plasmids for standards preparation
A region of 119 bp PCR fragment of TTV genome was amplified using primers TTVf and TTVr (Table 3). Resulting amplicon was purified using QIAquick PCR Purification kit, quantified by spectrophotometer and then cloned into the TA cloning vector. The resulting plasmid was transformed into One Shot TOP10F` competent cells according to instructions provided by the manufacturer. Twelve, isolated colonies of transformed competent cells from solid luria-bertani medium containing ampicillin (100 μg/mL) were subjected for TTV insert confirmation. Each individual colony was suspended separately into 3 mL of liquid luria-bertani medium containing 100 μg/mL of ampicillin for overnight in a shaking incubator at 37°C with a speed of 225 rpm. Following overnight incubation, plasmids purification was done using QIA prep Spin Miniprep kit according to manufacturer’s instructions. Restriction enzyme digestion with EcoRI for the purified plasmids was done to confirm the presence of cloned TTV insert (119 bp) on 1.5% agarose gel electrophoresis (data not shown). TTV insert (119 bp) cloned into TA vector were sequenced for all the 12 separate clones using M-13 forward and reverse primers and confirmed by aligning with the TTV sequence (Gen Bank acc. no. AB008394). This plasmid with TTV inserts was linearized with BamHI enzyme and then used for preparation of standards in serial 10 fold dilutions from 10×109 copies to 20 copies/μL.
Absolute quantification of TTV DNA
PCR reaction for absolute quantification of TTV DNA using SYBR Green in a 25 μL reaction is as follows: each reaction contained 12.5 μL SYBR Green PCR master mix, 5 μL of template (serial 10 fold dilutions of the linearized plasmid standards or/ extracted DNA from the plasma samples of healthy blood donors), 1.25 μL (500 nm) of each primer (TTVF-1, TTVF-2, TTVR-1, TTVR-2) and 2.5 μL of Milli-Q water. The cycling conditions included initial activation of AmpliTaq Gold DNA polymerase (present in SYBR Green master mix) for 10 minutes at 95°C. The subsequent PCR conditions consisted of 40 cycles of denaturation at 95°C for 15 seconds and annealing and extension at 60°C for 1 minute per cycle. After real-time data acquisition, the cycle threshold value was calculated by determining the point at which the fluorescence exceeds an arbitrary threshold limit. Standards with known TTV DNA copies were prepared in two independent serial dilutions and were run in the range of 100 copies to 10×109 copies on four non-consecutive days to evaluate biological, inter, intraday variability and accuracy of the assay. In addition, a series of standards from one serial dilution were also run in triplicates on two different days to evaluate the intra-day and inter-day variations. The variability of the assay was evaluated by comparing the CT values run on the same day (intra-day) and on different days (inter-day) and was represented as co-efficient of variations (CV). Accuracy was calculated by taking the ratio of back calculated copies from the standard curve to the theoretical copy number of the reactions. Real-time PCR assay for test samples (HSCT recipients) and for biological replicates of each healthy individual were performed with the inclusion of TTV plasmid standards and negative controls in each run. In addition to this, precision of the assay was also checked by running known TTV positive DNA (positive controls with exact log copies/mL). The viral genomic copies per mL of plasma was calculated as described by Huang et al.  i.e., by multiplying the copies per reaction by a factor of 30 [30 μL extracted DNA/5 μL of template x (1 mL/200 μL plasma)].
Melting curve analysis for specificity
Following amplification, melting curve or dissociation curve analysis was performed to measure the specificity of the PCR product. The temperature program used for the melting curve analysis was 95°C for 15 seconds followed by 60°C for 1 minute and then 95°C for 15 seconds with ramp rate of +0.3°C/second.
We are thankful to all the patients and healthy donors who consented to participate in this study. This investigation was supported by grants from CANSEARCH and the Hans Wilsdorf Foundations. We also would like to thank Professor Fabrizio Maggi Group (Retrovirus Center and Virology Section, Department of Biomedicine, University of Pisa, Pisa, Italy) for providing us the TTV positive DNA that were also used for checking the assay’s precision.
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