Objective: To establish a rapid specific method to identify the single-nucleotide polymorphisms (SNPs) of HBV polymerase gene region which are the methionine residue of the conserved YMDD motif.
Methods: Two specific primers were designed to amplify interested gene region involved in SNPs which were also used as HBV DNA identification. Specific primers of SNaPshot were designed to detect 741A-G (YVDD), 743G-T (YIDD). The different fluorescent dye labeled ddNTP was used to further extend the strand of PCR product and was detected by ABI PRISM 310 Genetic Analyzer. Sera from 13 patients with chronic hepatitis B after lamivudine treatment were analyzed.
Results: Aside from mutation of YMDD, there were mutations of 514C-A, 523C-A, 562T-A, 667C-A. The 13 samples were simultaneously tested with SNaPshot and DNA sequencing, the same results were obtained. The method of SNaPshot showed high specificity.
Conclusion: Mutation of YMDD results in the changes of ATG codon, and there are new ATG codon in the upper strand of YMDD. SNaPshot technique is rapid, specific and accurate for the SNPs monitoring of HBV DNA mutation during lamivudine therapy. Two samples were determined by SnaPshot technique, identifying the co-existence of the mixed wild type and mutant type HBV infection.