Total DNA in the supernatant was extracted with a TIAN amp Virus DNA/RNA Kit (Tiangen, China), and 2 μl of the extracted DNA solution was subjected to real-time PCR to quantify extracellular HBV virions. HBV replication intermediates in cells were extracted according to a previous study [20], and 2 μl of the extracted DNA solution was subjected to real-time PCR. Real-time PCR was carried out by using TB Green Premix Ex Taq II (TaKaRa) in a Step-OnePlus Real-Time PCR System (Thermo Fisher Scientific) using the primers 5′-GTTGCCCGTTTGTCCTCTAATTC and 5′-GGAGGGATACATAGAGGTTCCTT. PCR was performed with the following parameters for 40 cycles: 95 °C for 5 s and 60 °C for 30 s.

Total RNA was purified from cells using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. One-step real-time RT-PCR was performed with 100 ng of total RNA using a One-step RT-PCR kit (TaKaRa) on a Step-One Plus Real-Time PCR System (Thermo Fisher Scientific) using the primers 5′-CCGTCTGTGCCTTCTCATCT and 5′-TAATCTCCTCCCCCAACTCC to detect HBV RNA levels. RT-PCR was performed with the following parameters for 40 cycles: 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s.

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