中文摘要：

目的建立一种新的单管巢式实时荧光PCR熔解曲线法用于快速灵敏地筛检临床乙型肝炎病毒（HBV）样本核心启动子区nt1758-1777片段缺失突变。方法通过比对分析Gen Bank收录的HBV基因序列,设计通用巢式PCR引物,建立优化方法体系;对340例慢性乙型肝炎患者血清HBV CP区进行扩增,产物直接测序,并随机选择50例样本进行克隆测序,分析nt1758-1777缺失突变检出率。野生型和缺失型标准质粒来自于单克隆测序样本,并对两种标准质粒及克隆检测样本进行荧光定量PCR扩增,获得熔解曲线及熔解温度,得到熔解曲线的阳性标准。用新建立的方法对340份样本进行检测,并用焦磷酸测序加以验证。结果直接测序法检出16例（4.7%）阳性样本。标准质粒及克隆检测样本中缺失序列比例≥15%的样品,其熔解温度（Tm）≥88.3℃,遂将≥88.3℃作为新方法的阳性标准。用新方法检出47例（13.8%）阳性样本,其中用焦磷酸测序验证缺失突变比例≥1.0%的样本38例,〈1.0%的样本9例;15份阴性样本缺失突变比例均〈1.0%。用焦磷酸测序验证缺失突变比例1.0%作为阳性阈值,新方法对nt1758-1777缺失检测的阳性符合率为80.9%,阴性符合率为100%,两种方法的一致性Kappa值为0.671。结论与直接测序法相比,新方法可显著提高nt1758-1777缺失检出率,结合焦磷酸测序证实,可有效提高检测的准确性和经济性。该方法对建立HBV其他缺失突变的检测方法也可提供借鉴。

英文摘要：

Objective Nucleotide（nt） 1758-1777 deletion in core promoter（CP） region of hepatitis B virus（HBV） has been suggested to be associated with disease progression. However, the complicated and less sensitive assay for it limited its use in clinic. The present study was aimed at setting a novel assay for its detection using single-tube nested PCR amplification and real-time P CR melting curve analysis. Methods The PCR primers were designed through analysis of HBV genomic sequences in Gen Bank, and detection conditions were optimized. HBV CP region from 340 serum samples of chronic hepatitis B patients were amplified and directly sequenced, and fifty samples were randomly selected for cloning and sequencing for analysis of nt 1758-1777 deletion. The wild-type and deletion-type plasmids were extracted from mono-cloning samples. Positive standard of melting curve analysis was set up in light of the results of PCR amplification of two standard plasmids and cloning samples. The new method of assay was used in 340 samples, and the data were verified by the results of pyrosequencing. Results Sixteen（4.7%） samples were positive for the deletion by direct sequencing, and no less than 15% samples in standard plasmids and cloning sequencing showed sequence deletion. The melting temperature（Tm） of deletion-type plasmid and cloning samples containing ≥15% proportion of the deletion sequence was ≥88.3℃, which was determined as positive standard of the novel assay. Forty-seven（13.8%） samples were detected positive for nt 1758-1777 deletion by the novel assay. Among them, deletion ratio was ≥1.0% in 38 samples and 1.0% in 9 samples by pyrosequencing, respectively. The deletion ratio was all 1.0% in 15 negative control samples. The deletion ratio of 1.0% was taken as positive cutoff by pyrosequencing, the novel assay had 80.9% positive consistency and 100% negative consistency, with a Kappa value of 0.671. Conclusions Comparing with direct sequencing, the novel assay significantly increased detection ra