RFLP基因分型在动态监测乙型肝炎病毒基因变异中的应用

目的探讨基因分型结合克隆后测序方法在动态监测ＨＢＶ基因变异中的价值。方法分析１例重型肝炎患者发 病后血清，用 ＰＣＲ扩增血清中 ＨＢＶ ｐｒｅＳ／Ｓ基因片段并克隆，２份标本各随机挑取 ３个阳性克隆进行测序；同时用 ＲＦＬＰ方法分析２份血清中ＨＢＶ毒株的基因型。结果ＰＦＬＰ分析２份血清为Ｂ基因型为主的Ｂ／Ｃ基因型混合毒株感 染；６个克隆中仅１株为Ｃ基因型。比较２份血清ＨＢＶ ｐｒｅＳ／Ｓ克隆核酸序列，有１７０个点位的差异，剔除Ｃ基因型株 后，仅５４个位点差异。结论克隆后测序结合ＲＦＬＰ对ＨＢＶ基因型测定有助于更准确地对ＨＢＶ基因变异进行动态分 析。

Application of restriction fragment length polymorphism for genotyping in dynamic monitoring of genomic mutations of hepatitis B virus

Ohjectie To assess the value of hepatitis B virus genotyping by restriction fragment length polymorphism (RFLP) combining nucleotide sequencing of the cloned gene fragment of hepatitis B virus (HBV) in dynamic monitoring of HBV genomic mutations. Methods Two serum samples from a patient with fulminant hepatitis B before and after the the onset of the disease were obtained to amplify the preS and S gene of hepatitis B virus by PCR, the product of which was cloned into plasmid pGEM-T. Three positive clones were selected at random from each sample for sequencing, and RFLP analysis was performed to determine the HBV genotype of the 2 serum samples. Results Mixed infection with HBV strains of genotype B and C, with genotype B strains prevailing, was identified in the 2 serum samples. Only 1 cut of the 6 clones belonged to genotype C. Comparison of the preS and S genomic sequence of all the clones revealed 170 sites that differed, which was reduced to merely 54 sites when eliminating the genotype C clone. Conclusion RFLP analysis for HBV genotyping is useful in dynamic monitoring and analysis of HBV genomic variation.