2.6. Quantification of Serum HBV pgRNA

Serum HBV pgRNA levels were detected by qPCR on the LightCycler 480 II Real-Time PCR Detection System (Roche, Mannheim, Germany) by using a SYBR Green method. The primers used to detect 3.5 kb HBV pgRNA are provided in Supplementary Table S1. PCR was used to construct the standards by using each primer from the HBV full genome (accession number: {"type":"entrez-nucleotide","attrs":{"text":"KJ790199","term_id":"769114823","term_text":"KJ790199"}}KJ790199) [30], and the PCR products were subsequently ligated into the T&A™ Cloning Vector (Yeastern Biotech, New Taipei, Taiwan). The qPCR reaction mixture (20 μL) contained 10 μL of 2× GoTaq^® Green Master Mix (Promega Corp., Madison, WI, USA), 0.5 μL of the forward primer (10 μM), 0.5 μL of the reverse primer (10 μM), 1 μL of the cDNA template, and 8 μL of double distilled water (ddH₂O). The reaction mixture was denatured at 95 °C for 5 min, followed by 40 cycles at 95 °C for 20 s and 60 °C for 40 s. The LOD of serum HBV pgRNA was 1466 copies/mL, as calculated by probit analysis (Supplementary Table S2). For statistical analysis, 1465 copies/mL (3.17 log copies/mL) was recorded as the value for serum samples with HBV pgRNA below LOD or not detected.