The target gene fragments of HDV were synthesized and cloned into the pUC57 vector (Biomed Biotechnology, Beijing China). Synthetic DNA targets with a forward primer that contained a T7 promoter sequence were transcribed in vitro to generate synthetic RNA targets. The product was used as the template using the HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs, USA) at 37°C overnight.
The transcribed RNA was then treated with RNase-free DNase I (Qiagen, Germany) to remove any remaining DNA according to the manufacturer’s instructions. RNA was purified using RNA Clean XP (Beckman Coulter, USA), quantified by Nanodrop and Qubit and diluted in nuclease-free water to working concentrations. HDV RNA was extracted from 200 µl plasma samples using an automated nucleic acid extraction system (Bioteke, China) according to the manufacturer’s instructions.
According to previous studies, thermal shock was used to disrupt the secondary structure of HDV RNA, that is, 10 µL of HDV RNA at 95°C for 10 min, immediately followed by cooling to −80°C [22 (link),23 (link)].
The transcribed RNA was then treated with RNase-free DNase I (Qiagen, Germany) to remove any remaining DNA according to the manufacturer’s instructions. RNA was purified using RNA Clean XP (Beckman Coulter, USA), quantified by Nanodrop and Qubit and diluted in nuclease-free water to working concentrations. HDV RNA was extracted from 200 µl plasma samples using an automated nucleic acid extraction system (Bioteke, China) according to the manufacturer’s instructions.
According to previous studies, thermal shock was used to disrupt the secondary structure of HDV RNA, that is, 10 µL of HDV RNA at 95°C for 10 min, immediately followed by cooling to −80°C [22 (link),23 (link)].
