Qiaamp dna extraction kit

DNA was extracted from 200 μl of plasma using the Qiagen QIAamp® DNA extraction kit (QIAGEN® Inc., Hilden, Germany) according to the manufacturer’s instructions. Three pairs of primers previously described by Chekaraou et al. (2010) (link) were used to amplify 3 overlapping amplicons of 1,228-bp (nt 2,817–863), 1,253 bp (nt 448–1,701), and 1,653 bp (nt 1,609–80) covering the whole HBV genome as shown in Figure 1.
PCR reactions were performed in 50 μl of reaction mixture containing 1X polymerase buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.2 μM of each primer, 1.25 U of Taq Core MP® (Applied Biosystems) and nuclease-free water. The amount of DNA extract added varied between 10 to 35 μl depending on the viral load. PCR cycling was as follows: 94°C for 5 min, 40 cycles (94°C for 1 min, 56°C/57°C/62.5°C for regions 1, 2, and 3, respectively, 72°C for 1 min) with a final extension step at 72°C for 10 min. PCR products were analyzed by electrophoresis on 1% agarose gels stained with 1,25X of Red gel™ dye Nucleic Acid (Biotium®) and visualized by UV transilluminator.
The purified template DNA was sequenced using a BigDye Terminator Ready Reaction Cycle Sequencing Kit (Applied Biosystems) using the same primers pairs on an ABI Prism 3130 Genetic Analyzer (Applied Biosystems).

Genomic DNA was extracted from lymphocytes (phase 1) or buccal DNA (phase 2) using the Qiagen QIAamp DNA extraction kit (Qiagen Inc., Valencia, CA) and stored at − 80 °C. Telomere length (T) relative to a single copy gene 36B4 (S) was measured using a singleplex quantitative polymerase chain reaction (qPCR), as described by Cawthon [6 (link)]. All assays were run in triplicate and a standard laboratory control was run on each plate. Each plate run was then assessed for efficiency and precision (R2) using the standard curve generated using the laboratory control. R2 values for the telomere and control gene standard curves were greater than 0.98, demonstrating great precision. Efficiency for the telomere and control gene PCRs were between 99% and 110%. The mean normalized T/S ratio was used for the statistical analysis.

Viral DNA load within the hearts was evaluated by quantitative PCR (qPCR) at 3, 7, and 14 dpi. From each heart, 0.1 g tissue was homogenized, and DNA was isolated using the QiaAmp DNA extraction kit (Qiagen, Hilden, Germany). The early gene 1 (E1) was amplified by using E1-specific primers (F-5′TCGCCCATCGTTTCGAGA-3′), (R-5′TCTCGTAGGTCCACTGACGGA-3′), and probe (6FAM-ACTCGAGTCGGACGCTGCATCAGAAT-TAMRA), as previously described [15 (link),37 (link),38 (link)]. Viral copy number was determined using the pCB-E1 plasmid containing the MCMV early gene 1. E1 was cloned into a pcDNA3.1 plasmid by using the fast cloning technique [39 (link),40 (link)]. The qPCR assay was conducted using an AB7000 machine (Applied Biosystems, Foster City, CA, USA) with the following temperature profiles: 50 °C for 2 min; 95 °C for 10 min; and 40 cycles of 95 °C for 15 s, 60 °C for 1 min, and 70 °C for 1 min.