Viral RNA was extracted from the patients’ serum (200 μL stored at −80 °C) using NucleoMag 96 Virus (Macherey-Nagel, Düren, Germany) and automated KingFisher™ Magnetic Particle Processors (Thermo Fisher Scientific Inc., Waltham, MA, USA) in accordance with the manufacturer’s instructions. Serum samples from healthy subjects were used as negative controls. The RNA was eluted in 50 μL of nuclease-free distilled water, and reverse transcribed using the SuperScript III reverse transcriptase protocol (Thermo Fisher Scientific, Waltham, Massachusetts, USA): the cDNA was amplified by means of nested polymerase chain reaction (PCR) using GoTaq® DNA Polymerase (Promega, Madison, WI, USA). The primers for the first and second rounds of NS5B amplification and the PCR conditions have been previously described [42 (link),43 (link)].
The fragments obtained by means of PCR were purified using a commercial purification kit (QIAquick PCR Purification Kit, Qiagen, Hilden, Germany), and then sequenced bi-directionally using a BigDye Terminator Kit version 3.1 (Applied Biosystems, CA, USA) in accordance with the manufacturer’s instructions.
The sequencing products were purified from a 10 μL sample by means of precipitation in an ethanol/sodium acetate mixture. Finally, the sequences were determined using an automated DNA sequencer (ABI PRISM 3130 XL Genetic Analyser, Applied Biosystems).
The fragments obtained by means of PCR were purified using a commercial purification kit (QIAquick PCR Purification Kit, Qiagen, Hilden, Germany), and then sequenced bi-directionally using a BigDye Terminator Kit version 3.1 (Applied Biosystems, CA, USA) in accordance with the manufacturer’s instructions.
The sequencing products were purified from a 10 μL sample by means of precipitation in an ethanol/sodium acetate mixture. Finally, the sequences were determined using an automated DNA sequencer (ABI PRISM 3130 XL Genetic Analyser, Applied Biosystems).