Validation of SYBR Green based quantification assay for the detection of human Torque Teno virus titers from plasma.
Validation of SYBR Green based quantification assay for the detection of human Torque Teno virus titers from plasma.
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DOI：
10.1186/1743-422x-10-191
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发表时间：
2013-06-11
期刊：
影响因子：
4.8
通讯作者：
Ansari M
中科院分区：
文献类型：
--
作者：
Tyagi AK;Pradier A;Baumer O;Uppugunduri CR;Huezo-Diaz P;Posfay-Barbe KM;Roosnek E;Ansari M
定量滴度的普遍存在的病毒，如扭矩teno病毒（TTV），不引起临床症状，可能有助于评估个人的免疫状态。我们在此描述了基于SYBR Green的血浆样本TTV定量方法的验证。使用具有TTV特异性插入物的质粒制备标准品，并使用SYBR绿色方法进行TTV的绝对定量。在4个非连续日评估了该方法的准确度和精密度（日内和日间）。TTV还从20名健康志愿者和30名造血干细胞移植（HSCT）受者的血浆样品中定量。该试验具有专属性，并显示出令人满意的效率（82.2%，R2=0.99），定量限定义为每个反应100个拷贝。在100 - 1010拷贝/反应范围内，该测定具有良好的精密度（循环阈值（CT）的日间和日间变异系数< 4%）和准确度（100 ± 10%）。我们发现TTV载量范围为2.5 - 4.07 log拷贝/mL血浆，健康个体的CT（平均值± SD）为33.8 ± 1.77，HSCT受者的血浆TTV载量范围为2.06 - 8.49 log拷贝/mL血浆，CT（平均值± SD）为24.3 ± 1.04。基于SYBR Green的q-PCR检测结合了简单性和令人满意的灵敏度，并且可能适用于监测移植受体的免疫状态，其中TTV负荷随时间的推移可以作为人血浆样品中免疫重建的标志物。
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.