High pure pcr template preparation kit

From ticks used to monitor viral replication or from the progeny used to test vertical transmission, DNA was extracted from 200 µl of each crushed tick supernatant using the High Pure PCR Template Preparation Kit (Roche Life Science, Penzberg, Germany). Primers and probes detecting the VP72 ASFV gene and the tick beta-actin gene were used for duplex qPCR as described previously [2 ]. For absolute quantification, a plasmid encoding the VP72 and tick beta-actin genes was used at different dilutions to obtain a standard curve and determine the number of gene copies in each sample (Additional file 2: Figure S1).
The qPCR described above was used to assay dissected tick organs. Organs were lysed using tissue lysis buffer according to the manufacturer’s procedure (High Pure PCR Template Preparation Kit; Roche Life Science); DNA was extracted using the same kit (Roche Life Science). As we did not monitor ASFV replication in organs and just needed an estimation of the genome viral load in organs at a given time, amplification results were only expressed as the quantification cycle (Cq), i.e., the number of amplification cycles required for the fluorescent signal to cross the detection threshold. A low Cq indicates a large quantity of DNA, while a high Cq indicates a small quantity of DNA.

DNA was extracted from naked mole-rat and mice blood samples following the manufacturer’s protocol using a High Pure PCR Template Preparation Kit (Roche, Penzberg, Germany). Three tissues (lungs, muscles and kidneys) were dissected from Spalax and rats and immediately frozen and stored at −80 °C. Genomic DNA was extracted from these tissues using a High Pure PCR Template Preparation Kit (Roche, Germany). All DNA samples were tested for purity and integrity using a Nanophotometer NP80 (Implen, Munchen, Germany).

To eliminate free-floating DNA that exists outside of EVs, purified EVs were added to a microtube. Then, the EVs were treated with 10x reaction buffer (200 mM Tris-HCl, pH 8.3, 20 mM MgCl2) and DNase I (Sigma, St. Louis, MO) and then incubated for 15 min at room temperature. To bind calcium and magnesium ions and to inactivate the DNase I, the sample was treated with stop solution (50 mM EDTA), and the microtube was heated at 70 °C for 10 min to denature the DNase I and then chilled on ice. After elimination of free-floating DNA, the EVs were lysed by mixing cell lysis buffer and detergent, and the EV-derived DNA was purified using the High Pure PCR Template Preparation Kit (Roche Diagnostics, Mannheim, Germany). The volume of sample used for the isolation of cfDNA was 500 μL of pleural effusion supernatant. cfDNA was isolated from the supernatant of the pleural effusion using the High Pure PCR Template Preparation Kit (Roche Diagnostics, Mannheim, Germany). We eluted cfDNA with 50 μL of distilled water. The purification of DNA was measured using a NanoDrop Spectrophotometer (Thermo Fisher Scientific, Waltham, MA USA). The concentration and distribution of double-strand DNA was measured using a 4200 TapeStation (Agilent Technologies, Santa Clara, CA, USA).

Specimen manipulations and DNA extractions were performed in a class II biological safety cabinet. DNA from cultures and clinical specimens was extracted using either the NucliSens^® EasyMag^® (bioMérieux) or the High Pure PCR Template Preparation kit (Roche Diagnostics Australia Pty Ltd., North Ryde, Australia) according to the manufacturer’s instructions for the High Pure PCR Template Preparation kit, including the addition of MagNA Lyser Green Beads (Roche Diagnostics Australia Pty Ltd.), bead beating of tissue samples and a final elution volume of 80 µL for all specimen types [20 (link)]. DNA was stored at −20 °C prior to use.

Genomic DNA isolation was performed from fresh frozen and formalin-fixed, paraffin-embedded tissue biopsies using a High Pure PCR Template Preparation Kit (Roche) according to the manufacturer’s protocol.
Buffy coat layers were separated from whole blood specimens by centrifugation at 1350 rcf for 12 min. Genomic DNA was isolated from 200 µL buffy coat fractions (High Pure PCR Template Preparation Kit, Roche) in line with the manufacturer’s instructions. In the case of the Cell-Free DNA Collection Tube (Roche), ProtK digestion was performed for one and two hours.
Plasma fractions were separated from whole blood samples by two centrifugation steps (1350 rcf, 12 min). CfDNA isolation was carried out from 1–2 mL plasma samples with the Quick-cfDNA™ Serum & Plasma Kit (Zymo Research, Orange, FL, USA) according to the manufacturer’s recommendation. All DNA samples were stored at −20 °C until further examinations.
A NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, Orange, FL, USA) was applied to measure the concentration and purity (OD260/280, OD230/280) of both tissue and buffy coat genomic DNA, while quantification of cfDNA was carried out with a Qubit 1.0 fluorometer using a Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific).

Genomic DNA was prepared using the Roche High Pure PCR Template Preparation kit (Roche Diagnostics, Indianapolis, IN, USA). In brief, 200 μl whole blood, 200 μl Binding Buffer and 40 μl proteinase K were heated at 70 °C for 10 min. Then, 100 μl of isopropanol, 500 μl of Inhibitor Removal Buffer and 500 μl of Washing Buffer were added sequentially. After each addition, the solution was centrifuged at 8000 rpm for 1 min. A 50-μl aliquot of elution buffer was used to elute the DNA, and the extracted DNA was stored at − 20 °C until use. Aliquots of 1 μl DNA were used to run the nested PCR for identifying the five human Plasmodium species according to previously reported methods [21 (link), 22 (link)].

DNA was extracted from 200 μl of whole-blood samples using a Roche High Pure PCR Template Preparation Kit (Roche, Mannheim, Germany) in accordance with the manufacturer's instructions. The DNA samples were then stored at −20°C or directly used for analyzes. The FRET-qPCR targeting BLV pol gene was applied to screen BLV provirus, as described previously (sensibility: 1 copy/reaction) (Yang et al., 2016a (link)).
Plasma was separated from whole blood samples by centrifugation at 1,500 × g for 20 min. The plasma samples were then stored at −20°C or directly used for analysis. The commercial INgezim BLV Compac 2.0 blocking ELISA kit (Ingenasa, Madrid, Spain) was also used to detect the specific antibodies to BLV gp51 protein following the manufacturer's instructions.