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

作　　者：王美容[1] 邱宁[1] 卢实春[2] 修典荣[3] 于建国[4] 李彤[1] 刘学恩[1] 庄辉[1] 

机构地区：[1]北京大学医学部病原生物学系,100191 [2]首都医科大学附属佑安医院外科 [3]北京大学附属第三医院外科 [4]解放军第88医院

出　　处：《中华流行病学杂志》2011年第5期504-509,共6页Chinese Journal of Epidemiology

摘　　要：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HBVDNA（tDNA）和cccDNA特异性引物及探针，用3．44×10^0～3．44×10^9copies／pl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β—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3．44×10^0～3．44×10^9copies／μl。检测HBV cccDNA和HBVDNA下限均为3．44×10^0copies／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0．003copies／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7．24×10^2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。Objective To establish and optimize a sensitive and specific quantitative real- time 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 （3.44×10^0～3.44×10^9copies/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 × 10^0 to 3.44 × 10^9 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 × 10^0 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 0.031 copies/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 × 10^2 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

关 键 词：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 

分 类 号：R512.62[医药卫生—内科学]