Ttv r gene assay

TTV quantification was performed using the TTV R-Gene® assay (BioMérieux, Marcy-l’Etoile, France), a real-time polymerase chain reaction (PCR) assay targeting the TTV 5′ untranslated region (27 (link)). The assay has a dynamic range of 250 to 10⁹ copies/mL, with a limit of detection at 250 copies/mL. The assay was developed for quantifying TTV in plasma and whole blood samples, for which it is validated and widely used (28 (link)). We recently demonstrated that TTV load as quantified by the TTV R-Gene® assay did not differ significantly whether quantified in serum or plasma and observed a very strong and highly statistically significant correlation between serum and plasma TTV load, underscoring the interchangeability of serum and plasma for TTV quantification (21 (link)).
TTV DNA was extracted from serum samples using the QIAsymphony SP platform (QIAGEN, Venlo, the Netherlands), and PCR was conducted on a Light Cycler® 480 Instrument II (Roche Diagnostics, Rotkreuz, Switzerland) according to the manufacturer’s instructions. Viral load was determined using a standard curve, and specimens with undetectable viral load were assigned a value of 0.01 copies/mL for analysis purposes, as previously done by Fernández-Ruiz et al. (20 (link)).

Quantification of TTV-DNA viral load from whole blood was performed using the standardized TTV R-GENE® kit (bioMérieux, France), as specified by the manufacturer (40 (link)). Viral DNA was extracted from 200 μl EDTA whole blood using an easyMAG extractor (bioMérieux, France) with 140 μl of silica and a 50 μl elution volume. Ten μl of eluate was added to 15 μl of ready-to-use amplification premix (TTV R-GENE® assay, bioMérieux, France). In each run, an internal control and four quantification standards provided by the manufacturer were included. Amplification was performed according to manufacturer’s instructions on CFX96™ Real-Time PCR Detection System (Bio-rad Hercules, CA, USA). The limit of detection was 2.4 log₁₀ copies/ml and the quantification range was between 2.4 to 9 log₁₀ copies/ml.