摘要
目的:建立一种快速检测乙型肝炎病毒(HBV)准种遗传异质性的方法并验证其敏感性和特异性。以满足研究HBV准种需要检测大量病毒序列的要求。 方法:在构象敏感凝胶电泳(CSGE)检测方法的基础上,针对HBV核酸序列组成的特殊性,调整凝胶中使用的甲酰胺和乙二醇浓度并加入一定量的尿素,另外通过一次预电泳优选驱动序列。最后用改进地CSGE检测已知序列的HBV C-ORF区片段克隆,并把检测结果与DNA序列分析结果进行比对,验证该方法的敏感性和特异性。 结果:用改进地CSGE检测克隆的HVC C-ORF区片段获得了清楚地电泳图谱。对已知序列的HBC C-ORF区克隆片段进行检测:在34个克隆中发现了27种不同的克隆型,准种内病毒株(克隆)频率介于2.9～17.6％,克隆型之间的遗传距离介于0.2～2％,CSGE所得结果与DNA序列分析结果高度一致。 结论:我们建立的CSGE具有高度的敏感性和特异性,完全可以用于检测HBV准种序列的异质性及其他相关研究。
AIM: To establish a method for detection of HBV quesispecies heterogeneity and to determine its sensibility and specificity.METHODS: Base on the conformation-sensitive gel electrophoresis(CSGE)of HBV gene sequences, the authors changed the concentration of formamide and ethylene glycol and put urea in gel. Moreover, a preliminary CSGE was performed for selection of an optimal driver in each sample. Finally, 34 previously identified sequence plasmids that contained HBV C-ORF region were detected by CSGE with the improved conditions, and the results of CSGE were compared with that of nucleotide sequencing.RESULTS: The clear maps of CSGE were obtained by the improved method. In all 34 plasmids detected, the total numbers of distinct HBV C-ORF segment gel shift clonotypes were 27. The frequency of virus strains was 2.9-17.6 %. The genetic diversity of HBV quasispecies was 0.2-2.0%. The results of detection of CSGE coincided with that of nucleotide sequencing.CONCLUSION:The method for detection of hepatitis B virus quesispecies heterogeneity is highly sensitive and specific.
出处
《世界华人消化杂志》
CAS
2002年第4期411-414,共4页
World Chinese Journal of Digestology
