应用滚环扩增技术检测乙型肝炎病毒共价闭合环状DNA的研究

The study on the rolling circle amplification for detecting hepatitis B virus covalently closed circular DNA

目的 应用滚环扩增（RCA）技术检测HBV共价闭合环状DNA（cccDNA）,观察该方法的特异性和敏感性.方法 以HBV全基因质粒为模板,经酶切、连接、浓缩、胶回收纯化等步骤,构建和制备HBV cccDNA标准品.抽提7例慢性乙型肝炎患者肝组织总DNA,进行滚环扩增.用HBV cccDNA标准品、3.2 kb线性HBV DNA、健康肝组织总DNA及15例慢性乙型肝炎患者血清总DNA作为对照,验证此方法的特异性.将HBV cccDNA标准品进行系列稀释,了解该方法的敏感性.结果 成功构建了HBV cccDNA,并可用RCA方法进行扩增.RCA方法可从2 mg的慢性乙型肝炎患者肝组织中检测到HBV cccDNA,并可检测低至1×102拷贝/μL的HBV cccDNA.RCA方法不能检测3.2 kb线性HBV DNA,在健康肝组织及15例慢性乙型肝炎患者血清中亦检测不到HBV cccDNA.结论 RCA方法操作较方便,具有很高的特异性和敏感性.

Objective To set up the rolling circle amplification （RCA） system for detecting hepatitis B virus （HBV） covalently closed circular DNA （cccDNA）, and to evaluate the specificity and sensitivity of this system. Methods Plasmids containing full-length of wild-type HBV genome were treated with restriction enzyme and T4 DNA ligase, and then were concentrated. The DNA fragments were recovered by the nucleic acid purification kit and severed as standard HBV cccDNA. Total DNA was extracted from hepatic tissues of seven chronic hepatitis B patients. RCA method was used to amplify genomes from tissue samples. Standard HBV cccDNA, 3.2 kb liner HBV DNA, normal hepatic tissue samples and 15 serum samples of patients with chronic HBV infection were used as controls to determine the specificity of RCA. Ten-fold serial dilutions of standard HBV cccDNA were used for determining the sensitivity. Results The standard HBV cccDNA was successfully constructed and could be detected by RCA method. HBV cccDNA could be amplified from 2 mg hepatic tissue samples at least of HBV infected patients, and could be detected as low as 1 ×102 copy/μL. cccDNA was not detected in 3.2 kb liner HBV DNA, normal hepatic tissue samples and 15 serum samples of chronic HBV infected patients. Conclusion RCA method can be used for rapid and simple detection of HBV cccDNA with high specificity and sensitivity.