A New Assay Based On Single Nucleotide Extension To Detect Point Mutation Of Hepatitis B Virus And Its Clinical Application

This research is composed of three parts: I . A new assay based on single nucleotide extension to detect point mutation of hepatitis B virus. II. The HBV YMDD mutation in sera of lamivudine-resisted patients and its assocciation with the quantification of HBV DNA. III. Detection of nt1896 point mutation in HBV precore region and clinical significance.I . A New Assay Based on Single Nucleotide Extension to Detect Point Mutation of Hepatitis B Virusobjective To develop a cheaper, faster assay for detecting point mutation in YMDD motif or pre-C gene of hepatitis B virus (HBV) and analyzing the percentage of each mutant strain. Methods The fragment which contains the point mutation was amplified by nested PCR. The positive strand of final PCR product was biotin-labelled by a 5'-biotin-labelled primer. PCR product of each sample was captured in four strepavidin coated microplate wells, then a detecting probe was hybridized to the captured positive strand of PCR product, the 3'end of which was designed to precisely overlap the mutant site. DNA polymerase and four kinds of fluorescin-12-ddNTP (A, U, C and G) were added in each well respectively. A single nucleotide will extend if the added fluorescein-12-ddNTP is complementary to the mutant site and will be detected with a peroxidase conjugated anti-fluorescein and TMB substrate. Results Single nucleotide extension assay can be completed in about five hours. We analysed HBV YMDD motif mutation of 57 samples by the single nucleotide extension assay, and All of the mutant sites of the 57 samples detected are completely in accord with the results of the commercial sequencing. Experiments with a series of different proportionally mixed wild and mutant standard PCR products showed this assay can effectively detect as little as 10%mutant strain in mixture. Conclusion The single nucleotide extension assay is a convenient, fast method to detect point mutation with high sensitivity and specificity, and it can determine the percentage of each virus strain in mixture.