| 王美容,邱宁,卢实春,修典荣,于建国,李彤,刘学恩,庄辉.肝组织中HBV cccDNA荧光定量聚合酶链反应检测法的建立[J].Chinese journal of Epidemiology,2011,32(5):504-509 |
| 肝组织中HBV cccDNA荧光定量聚合酶链反应检测法的建立 |
| A quantitative real time polymerase chain reaction for detection of HBV covalently closed circular DNA in livers of the HBV infected patient |
| Received:December 15, 2010 |
| DOI： |
| KeyWord: 乙型肝炎病毒 共价闭合环状DNA(cccDNA) 实时荧光定量聚合酶链反应 |
| English Key Word: Hepatitis B virus HBV DNA Covalently dosed circular DNA Real-time PCR |
| FundProject:"十一五"国家科技重大专项(2009ZX10004-314,2008ZX10002-004) |
| Author Name | Affiliation | E-mail | | WANG Mei-rong | Department of Microbiology, Peking University Health Science Center, Beijing 100191, China | | | QIU Ning | Department of Microbiology, Peking University Health Science Center, Beijing 100191, China | | | LU Shi-chun | Department of Hepatobiliary Surgery & Liver Transplantation Center, Beijing Youan Hospital, Capital Medical University | | | XIU Dian-rong | Department of Surgery, Peking University Third Hospital | | | YU Jian-guo | The 88th Hospital of People's Liberation Army | | | LI Tong | Department of Microbiology, Peking University Health Science Center, Beijing 100191, China | | | LIU Xue-en | Department of Microbiology, Peking University Health Science Center, Beijing 100191, China | xueenliu@bjmu.edu.cn | | ZHUANG Hui | Department of Microbiology, Peking University Health Science Center, Beijing 100191, China | zhuangbmu@126.com |
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| Abstract: |
| 目的 建立和优化一种灵敏、特异的检测肝组织中乙型肝炎(乙肝)病毒(HBV)共价闭合环状DNA(cccDNA)荧光定量聚合酶链反应(荧光定量PCR).方法 设计检测HBV DNA(tDNA)和cccDNA特异性引物及探针,用3.44×100-3.44×109copies/μl含HBV全基因组序列(C基因型)质粒作为标准品,建立荧光定量PCR检测标准曲线.取33例乙肝肝癌患者肝组织标本、13例慢性乙肝患者肝活检组织标本和10例非乙肝患者肝组织标本,验证该法灵敏度和特异度.提取肝组织中DNA,取一部分进行质粒安全ATP依赖的DNA酶(PSAD)酶切;另一部分不酶切作为tDNA和β-globin检测样本,分别进行HBV cccDNA、tDNA和p-globin定量检测,以β-globin为参比,对每个细胞的HBV cccDNA和tDNA含量进行标准化.结果 检测肝组织中HBV cccDNA和tDNA定量的线型范围均为3.44 × 100-3.44×109 copies/μl.检测HBV cccDNA和HBV DNA下限均为3.44×100copies/μl.33例乙肝肝癌患者和13例慢性乙肝患者的肝组织中HBV cccDNA最低含量分别为0.003 copies/cell和0.031 copies/cell;检测10份非乙肝肝癌患者的肝组织标本均为阴性.应用PSAD消化肝组织中提取的DNA可减少假阳性,提高cccDNA检测法特异度达7.24× 102倍.对2例乙肝肝癌患者的肝组织标本重复检测5次,Ct值的变异系数为0.224%～0.609%.结论 该方法灵敏度和特异度高,重复性好,可用于检测肝组织中HBV cccDNA. |
| English Abstract: |
| Objective To establish and optimize a sensitive and specific quantitative realtime polymerase chain reaction(PCR)method for detection of hepatitis B virus covalently closed circular DNA(HBV cccDNA)in liver tissue. Methods Specific primers and probes were designed to detect HBV DNA(tDNA)and cccDNA. A series of plasmids(3.44 × 100-3.44 × 109 copies/μl)containing a full double-stranded copies of HBV genome(genotype C)were used to establish the standard curve of real-time PCR. Liver samples of 33 patients with HBV related hepatocellular carcinoma(HCC), 13 Chronic hepatitis B patients(CHB)and 10 non-HBV patients were collected to verify the sensitivity and specificity of the assay. A fraction of extracted DNA was digested with a Plasmid-Safe ATP-dependent Dnase(PSAD)for HBV cccDNA detection and the remaining was used for tDNA and β-globin detection. The amount(copies/cell)of HBV cccDNA and tDNA were measured by a real-time PCR, using β-globin housekeeping gene as a quantitation standard. Results The standard curves of real-time PCR with a linear range of 3.44 × 100 to 3.44 × 109 copies/μl were established for detecting HBV cccDNA and tDNA, and both of the lowest detection limits of HBV cccDNA and tDNA were 3.44 × 100 copies/μl. The lowest quantitation levels of HBV cccDNA in liver tissues tested in 33 HBV related HCC patients and 13 CHB patients were 0.003 copies/cell and 0.031copies/cell, respectively. HBV cccDNA and tDNA in liver tissue of 10 non-HBV patient appeared to be negative. The true positive rate was increasing through the digestion of HBV DNA by PSAD, and the analytic specificity of cccDNA detection improved by 7.24 × 102 times. Liver tissues of 2 patients were retested 5 times in the PCR for detecting cccDNA and the coefficience of variations on cycle threshold (Ct)were between 0.224%-0.609%. Conclusion A highly sensitive and specific quantitative real time PCR method for the detection of HBV cccDNA in liver tissue was established and could be used for clinical and epidemiological studies. |
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