a novel assay for detecting the mutation of nucleotide 1758-1777 deletion in core promoter region of hepatitis b virus

Abstract

Objective 　Nucleotide (nt) 1758-1777 deletion in core promoter (CP) region of hepatitis B virus (HBV) has been suggested to be associated with disease progression. However, the complicated and less sensitive assay for it limited its use in clinic. The present study was aimed at setting a novel assay for its detection using single-tube nested PCR amplification and real-time PCR melting curve analysis. Methods 　The PCR primers were designed through analysis of HBV genomic sequences in GenBank, and detection conditions were optimized. HBV CP region from 340 serum samples of chronic hepatitis B patients were amplified and directly sequenced, and fifty samples were randomly selected for cloning and sequencing for analysis of nt 1758-1777 deletion. The wild-type and deletion-type plasmids were extracted from mono-cloning samples. Positive standard of melting curve analysis was set up in light of the results of PCR amplification of two standard plasmids and cloning samples. The new method of assay was used in 340 samples, and the data were verified by the results of pyrosequencing. Results 　Sixteen (4.7%) samples were positive for the deletion by direct sequencing, and no less than 15% samples in standard plasmids and cloning sequencing showed sequence deletion. The melting temperature (Tm) of deletion-type plasmid and cloning samples containing ≥15% proportion of the deletion sequence was ≥88.3℃, which was determined as positive standard of the novel assay. Forty-seven (13.8%) samples were detected positive for nt 1758-1777 deletion by the novel assay. Among them, deletion ratio was ≥1.0% in 38 samples and <1.0% in 9 samples by pyrosequencing, respectively. The deletion ratio was all <1.0% in 15 negative control samples. The deletion ratio of 1.0% was taken as positive cutoff by pyrosequencing, the novel assay had 80.9% positive consistency and 100% negative consistency, with a Kappa value of 0.671. Conclusions　Comparing with direct sequencing, the novel assay significantly increased detection rate of nt 1758-1777 deletion. In combination with pyrosequencing for confirmation, the accuracy and cost-effectiveness of the detection could be significantly improved. The novel assay offers an example for detecting HBV genetic deletions. DOI: 10.11855/j.issn.0577-7402.2016.03.04