HepG2.2.15细胞内乙型肝炎病毒cccDNA的定量检测  

Quantitative detection of hepatitis B virus cccDNA in infected hepatocytes

作　　者：赵克开[1] 缪晓辉[1] 徐文胜[1] 

机构地区：[1]第二军医大学附属长征医院感染科,上海200003

出　　处：《中华传染病杂志》2005年第1期6-9,共4页Chinese Journal of Infectious Diseases

基　　金：国家自然科学基金资助项目(300709580)

摘　　要：目的　建立一种细胞内乙型肝炎病毒cccDNA的定量检测方法。方法　消化收集处于对数生长期的HepG2.2.15细胞,取1×106 个细胞用小量质粒抽提试剂盒抽提细胞内的cccDNA,抽提产物用绿豆核酸酶酶切纯化,所得酶切产物用特异的引物和探针进行选择性荧光定量聚合酶链反应(PCR)检测。用对数生长期前的细胞培养上清液、4 份HBV DNA阳性和2 份HBV DNA阴性的慢性乙型肝炎(轻度)患者血清验证荧光定量PCR法的特异性,并用不同浓度的质粒标准品检测该方法的敏感性。结果　证实HepG2.2.15细胞内存在cccDNA,其含量约为每个细胞18 拷贝。对数生长期前培养上清液和慢性乙型肝炎(轻度)患者血清均未检测到荧光信号,本实验条件下用该方法可检测低至103 拷贝/ml的cccDNA分子。结论　该方法操作方便,特异性高,敏感性较好,可用于定量检测细胞内的cccDNA及抗病毒药物的筛选和评价等。Objective To establish a method for quantitative detection of hepatitis B virus covalently closed circular DNA(HBV cccDNA ) in infected cells. Methods The transfected cell line HepG2.2.15 which can consistently produce Dane particles was maintained in DMEM containing 380 μg/ml G418 and 10% fetal bovine serum. Cells in the exponential period were harvested from flasks, then intracellular HBV cccDNA was extracted from pellet containing 1×10~6 cells with mini plasmid extraction kit (QIAGEN).The extraction product was further purified by mung bean nuclease to remove HBV relaxed circular DNA possibly remained. HBV cccDNA was quantitatively detected by fluorescent PCR with selective primer set and Taqman MGB probe. Culture medium before exponential period, HBV DNA positive and negative serum samples from patients with chronic hepatitis B (mild) were amplified simultaneously to test the specificity of the fluorescent PCR method. Plasmids containing whole HBV genome were amplified with the same primer set and fluorescent probe to determine the sensitivity of the method. Results HBV cccDNA was detectable in HepG2.2.15, and the average quantity was 18 copies per cell approximately. No detectable fluorescent signal was observed when culture and serum samples were amplified. The detectable HBV cccDNA was as low as 10~3 copies per ml at least by this method. Conclusions This method is convenient, highly specific and highly sensitive. It can be utilized in the quantitative detection of intracellular HBV cccDNA as well as in the screening and evaluation of antiviral agents.

关 键 词：HEPG2.2.15细胞 定量检测 DNA 乙型肝炎病毒 细胞内 轻度 慢性乙型肝炎 方法 目的 阴性 

分 类 号：R446.5[医药卫生—诊断学]