Magna pure 96 dna

DNA was extracted from PBMCs using the MagNA Pure 96 DNA and the Viral NA Small Volume Kit (Roche, Basel, Switzerland). Cellular EBV load was then determined using quantitative real-time PCR. Samples that yielded > 100 viral copies/ul were selected for EBV genome sequencing.
We used the previously described enrichment procedure to increase the relative abundance of EBV compared to host DNA^{41 (link)}. Shortly, baits covering the EBV type 1 and 2 reference genomes were used to selectively capture viral DNA according to the SureSelect Illumina paired-end sequencing library protocol. Samples were then multiplexed and sequenced on an Illumina NextSeq sequencer^{41 (link)}.

RNA was extracted from patient samples and culture supernatants using MagNA Pure 96 DNA and Viral RNA Small Volume kits^® on a MagNA Pure 96TM instrument (Roche, France). ZIKV RNA was detected by amplification of an NS5 fragment (Real Star Zika virus RT-PCR kit 1.0, Altona Diagnostic GmbH, Germany) using a Light Cycler 480 instrument (Roche Molecular Systems). The limit of detection was 150 copies/mL. Both assays were performed according to the manufacturer’s instructions. Internal standards were used to correct for potential variation in the amount of input material. Quantitative RT-PCR values were further normalized and are given as viral RNA copy numbers/mL. Viral stocks were titrated using plaque-forming assay. Kinetic of viral production was determined as RNA copy number per mL, calculated based on a standard curve.

To quantify viral shedding, oropharyngeal and cloacal swabs were collected from the birds at 2, 4, 6, 8, and 12 days post-inoculation (dpi) and suspended in 1 mL of phosphate-buffered saline (PBS) containing antibiotics (400 mg/mL of gentamicin). Viral RNA was extracted from 200 µL of the suspension using MagNA Pure 96 DNA and Viral NA Small Volume Kit on the MagNA Pure 96 instrument (Roche Applied Sciences, Penzberg, Germany) according to the manufacturer’s instructions. Quantitative analysis of viral RNA was conducted using the cycle threshold (Ct) value using rRT-PCR targeting the M gene [43 (link)]. It has been demonstrated that Ct values correlate strongly with infectious viral titer [46 (link)]. To convert the Ct value into the EID₅₀ equivalent unit, we generated a standard curve between them as previously described [36 (link)]. Briefly, the HPAI H5N6 challenge virus with a known EID₅₀ titer was serially diluted 10-fold and quantified using rRT-PCR as described above. Ct values of viral 10-fold dilutions were plotted against viral EID₅₀ titers. The resulting standard curve showed a high correlation (r² > 0.99). The detection limit was 10² EID₅₀ equivalent/mL. Virus titers in samples were calculated by extrapolation of the standard curve equation.

Viral RNA or DNA was extracted from 140 μl of sample medium using the MagNA Pure 96 Instrument (Roche Life Science, Basel, Switzerland) according to the manufacturer's instructions (Magna Pure 96 DNA and RAN NA small volume kit).

DNA for the Leishmania infantum PCR and PARR analyses was extracted from the smears prepared for the lymph node cytology. Cell material was rubbed from the smears with a swab saturated by a phosphate-buffered saline (PBS) solution. Subsequently, the DNA was extracted from this swab using a commercial kit MagNA Pure 96 DNA and Viral NA Small Volume Kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturers’ instructions.