Abstract:

Background and purpose: It was reported that G1896A and G1899A mutation in the hepatitis B virus (HBV) precore region were all significantly associated with hepatocellular carcinoma (HCC). Simple, sensitive and reliable methods to detect precore mutations are needed in order to prevent the occurrence of HCC in clinical detection. The aim of this study was to develop a simple and sensitive reverse hybridization method for the detection of HBV precore mutation in HBV carriers. This method was applied for exploring the relationship between the precore mutations and HCC in patients of Qidong, Jiangsu Province. Methods: The specific probes of nt.1896 and nt.1899 were designed and synthesized. In order to improve the sensitivity and specificity, reaction conditions of reverse hybridization were optimized. We used reverse hybridization to detect 50 HCC serum samples and compared the results with direct sequencing. Then we used this method to assess the association between HBV precore mutations and HCC in serum samples from 50 HCC patients and 50 non-HCC controls. Results: The detection limit of reverse hybridization for HBV DNA level was 103 copy/mL and the sensitivity was 10% within a mixed virus population. The coincidence rate of reverse hybridization analysis was 98% compared with the direct sequencing results. It was showed that G1899A mutation was significantly associated with HCC compared to non-HCC controls (P=0.000, OR=4.846, 95%CI: 2.240-10.485), while G1896A mutation was not associated with HCC. Conclusion: Reverse hybridization is a simple and accurate approach for the detection of low amounts of HBV precore mutants among a mixed viral population. It has potential usage in the large-scale screening of precore mutations in chronic hepatitis B carriers.

Key words: Hepatitis B virus, Precore domain, Reverse hybridization, Mutation, Hepatocellular carcinoma