2.1. Sample Preparation

Blood samples were collected from 28 CHB patients treated with LAM at the Beijing Friendship Hospital from 2009–2016. Patients treated with LAM with primary non-response (defined as less than 1 log10 IU/mL decrease in HBV DNA level from baseline at three months of therapy) and poor response (defined as a decrease in HBV DNA of more than 1 log10 IU/mL, but detectable HBV DNA by real-time PCR assay at 24 weeks of treatment) were included in the study. Exclusion criteria were as follows: co-infection with other hepatitis virus (es), and patient who had poor compliance during treatment. The serum samples were detected via sequencing. Primers were designed for the mutant gene of HBV polymerase M204I, which were confirmed by the serum samples containing M204I mutation previously detected by sequencing (performed by TIANYI HUIYUAN). The upstream and downstream primer sequences were 5′-TTGGCTTTCAGTTATATGGATGAT-3′ and 5′-TAAAAAGGGACTCAAGATGCTG-3′, respectively. The capture sequence was NH₂-5′-TTTTTTTTTTTAAAAAGGGACTCAAGATGCTGTACAGACTTGGCC-3′, which was used to hybridize the amplified HBV DNA. Additionally, the sequence of the biotin-labeled DNA was biotin-3′-TTTTTTTTTTTCAGTTATATGGATTAT.