乙型肝炎病毒全基因组PCR扩增产物的直接测序

目的建立乙型肝炎病毒全基因组PCR扩增产物直接测序的方法并对序列进行分析。方法PCR法扩增乙型肝炎病毒全长基因，PCR扩增产物纯化后直接进行DNA序列测定；将已测定序列的PCR扩增产物用上述方法进行再测序以评价该方法的保真性；应用进化树分析及对齐比较等方法分析HBV基因型和YMDD变异情况。结果20例标本中有18例扩增阳性并得到全长基因组序列；保真性分析表明，本法引起的人为核苷酸突变率约为1‰；序列分析表明，18例标本中有10例为基因型B、8例为基因型C；2例出现耐拉米夫定的YMDD变异，均为基因型C。结论乙型肝炎病毒全基因组PCR扩增产物直接测序，方法简便，可满足临床和科研的需要。

Direct sequencing of PCR amplification products of Hepatitis B virus complete genome

Objective To establish a method for direct sequencing the PCR amplification products of complete genome of hepatitis B virus and analyse the sequences. Methods The complete genome of HBV were amplified by PCR. PCR products were sequenced directly. PCR products were resequenced to evaluate the fidelity of the method. The genotype and YMDD mutation of HBV were determined by phylogenetic analysis and alignment. Results Eighteen complete genome of HBV were amplified by PCR from 20 samples and the genome were sequenced; the fidelity analysis showed that artificial mutation rate of the method was 1‰. In 18 samples which HBV complete genome were amplified, 10 cases were genotype B, 8 cases were geno-type C, 2 cases were YMDD mutation and both were genotype C. Conclusions The PCR amplification products of HBV complete genome can be sequenced directly, this method is simple and convenient which can be used in sequence analysis of HBV complete genome on a large application.