CAJ | 학술논문

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.
목적 응용곤배확증(RCA)기술검측HBV공개폐합배상DNA(cccDNA),관찰해방법적특이성화민감성.방법 이HBV전기인질립위모판,경매절、련접、농축、효회수순화등보취,구건화제비HBV cccDNA표준품.추제7례만성을형간염환자간조직총DNA,진행곤배확증.용HBV cccDNA표준품、3.2 kb선성HBV DNA、건강간조직총DNA급15례만성을형간염환자혈청총DNA작위대조,험증차방법적특이성.장HBV cccDNA표준품진행계렬희석,료해해방법적민감성.결과 성공구건료HBV cccDNA,병가용RCA방법진행확증.RCA방법가종2 mg적만성을형간염환자간조직중검측도HBV cccDNA,병가검측저지1×102고패/μL적HBV cccDNA.RCA방법불능검측3.2 kb선성HBV DNA,재건강간조직급15례만성을형간염환자혈청중역검측불도HBV cccDNA.결론 RCA방법조작교방편,구유흔고적특이성화민감성.
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.