慢性乙型肝炎患者HBV cccDNA的检测

Detection of HBV cccDNA in chronic hepatitis B patients

目的建立和优化肝细胞内乙型肝炎病毒(HBV)共价闭合环状DNA(cccDNA)的检测方法。方法蛋白酶K裂解法释放肝细胞核内HBV cccDNA及基因组DNA,细胞裂解液进行核酸纯化后用酚-氯仿法抽提,并采用酶消化和设计特异性引物两种方法排除其他病毒复制中间体对检测结果的干扰,最后进行聚合酶链式反应(PCR)。结果以HBV松弛环状DNA(rcDNA)、阴性肝细胞、阴性血清作为对照,以包含HBV基因组全序列的质粒作为阳性对照,HBV感染者肝组织的PCR扩增产物进行琼脂糖凝胶电泳后出现的条带与目标片段分子量相等,且产物的DNA测序结果显示其序列与目标序列一致。外周血HBeAg(+)患者组肝组织检测结果的阳性率高于HBeAg(-)患者组。结论该方法检测肝细胞内cccDNA具有较高的特异性、敏感性,且成本较低。本方法临床标本初步检测结果显示病毒复制活跃患者肝组织中HBV cccDNA的阳性率较复制低的患者为高。

Objective To establish a sensitive specific and simplic method for detecting HBV cccDNA in hepatocytes of chronic hepatitis B patients. Methods Proteinase K was used for releasing HBV cccDNA and genomic DNA, then dividing the cell lysis solution into two parts , one for detecting HBV cccDNA , the other for detecting the number of β-globin as internal control. Nucleic acid for detecting HBV cccDNA extracted by phenol-chloroform was digested by plasmid-safe ATP dependent DNase,which was applied to digest the single strand DNA in RC DNA and ssDNA, and then was amplified by the selective primers using polyrnerase chain reaction（PCR）. The products were analyzed by agarose electrophoresis. Results HBV RC DNA, normal serum and liver biopsies were served as negative control, and the plasmid containing whole HBV genomic sequence was served as positive control. The PCR product of chronic hepatitis B patients analyzed by agarose electrophoresis was proved to be specific because its molecular weight was consistent with the goal fragment. The specificity of PCR product was further proved by gene sequencing. The positive rate of HBV cccDNA in HBeAg positive chronic hepatitis B patients was higher than that of HBeAg negative patients. Conclusion The method established in this study for detecting HBV cccDNA was more specific , sensitive and economical. The positive rate of HBV cccDNA was higher when virus replication was active.