RT-PCR Detection of Viral RNA from Serum Samples

Viral RNA was obtained from serum samples using a commercial kit (QIAamp Viral RNA Mini Kit, QIAGEN). Then, the cDNA was synthesized using the primer 1302D [22 (link)] 0.67 µM and the enzyme RevertAid RT (Thermo Scientific) following the suggested protocol. Amplification conditions and cycling were modified from protocols previously published [22 (link)]. The first round was carried out with 1× buffer, 2 mM MgCl₂, 0.2 mM dNTPs (Promega), 0.5 µM using the primers 8531U, and 1302D [22 (link)] (Supplementary Table S1) and Taq DNA polymerase (Thermo Scientific) 1.25. There were cycles of 95 °C for 2 min, followed by 35 cycles of 95 °C for 30 s, 56 °C for 30 s, extension at 72 °C for 1 min, and 72 °C for 10 min. The second round reaction was carried out with 1× buffer, 2 mM MgCl₂, 0.2 mM dNTPs (Promega), using the primers HDV-E and HDV-A at 0.5 µM [22 (link)], and Taq DNA polymerase (Thermo Scientific) 1.25. There were cycles of 95 °C for 2 min, followed by 35 cycles of 95 °C for 30 s, 56 °C for 30 s, extension at 72 °C for 1 min and 72 °C for 10 min.