慢性HBV感染者肝组织HBV cccDNA的定量检测  

Quantitative detection of hepatitis B virus cccDNA in chronic hepatitis B patients

作　　者：李军[1] 宋培新[1] 韩亚萍[1] 刘婷[1] 黄祖瑚[1] 

机构地区：[1]南京医科大学第一附属医院感染病科,210029

出　　处：《中华检验医学杂志》2007年第6期625-630,共6页Chinese Journal of Laboratory Medicine

基　　金：江苏省医学重点学科工程资助课题(135-07);江苏省卫生厅医学科技发展基金(H200311);江苏省"333工程"培养资金资助项目(2002)

摘　　要：目的建立一种肝活检组织中 HBV 共价闭合环状 DNA(cccDNA)的定量检测方法。方法待检肝组织标本共21份,来源于江苏省人民医院肝脏手术患者,包括19份慢性 HBV 感染,其中 HBeAg(+)标本10份,HBeAg(-)标本9份,4份非 HBV 感染为阴性对照组,取 HBV DNA 阳性的患者外周血作为 rcDNA 组。检测方法的主要步骤为肝组织经蛋白酶 K 和细胞裂解缓冲液消化后,用液相抽提法提取核酸,将提取的核酸溶液分为2等份。1份用特异性降解单链 DNA 的核酸酶加以消化,纯化后使用跨缺口引物和 SYBR Green Ⅰ荧光染料进行实时荧光定量 PCR 分析;另1等份则用以-定量检测肝细胞看家基因(β-Globin)作为样本细胞参数。检测结果的特异性主要通过 PCR 反应产物的序列分析及 rcDNA 组结果的对照进行证实,HBeAg(+)组和 HBeAg(-)组 cccDNA 水平的差异通过两样本 t 检验进行分析。结果 PCR 产物经琼脂糖凝胶电泳分析显示扩增产物的碱基数为350 bp左右,DNA 测序分析提示产物与目的片段的序列符合率为99%以上,且以 rcDNA 为对照的结果均为阴性,排除最有可能造成假阳性的 rcDNA 对结果的干扰。本方法对10 mg HBeAg(+)肝组织标本的cccDNA 的检测阳性率为100%。血清 HBeAg(+)的肝组织样本的平均 HBV cccDNA 水平高于HBeAb(+)的肝组织标本(P<0.05)。结论通过上述三种途径证实了本文所建立方法的特异性。应用 SYBR Green Ⅰ荧光染料和β-Globin 作为样本细胞参数所建立的实时荧光定量 PCR 方法检测肝细胞内 HBV cccDNA,具有较高的特异性、敏感性,且成本较低的特点。Objective To establish a method for detecting HBV cccDNA in hepatocytes of chronic hepatitis B patients. Method 21 liver biopsies from the hepatic operation patients in the hospital of jiangsu province, concluding 19 HBV chronic infected patients （10 HBeAg positive patients and 9 HBeAg negative patients） and 4 uninfected patients, HBV DNA（ ＋ ） serum of hepatitis B patients was thought as rcDNA. To use proteinase K to release HBV cccDNA and genomic DNA, then divide the cell lysis solution into two parts, one for detecting HBV cccDNA , the other for detecting the number of β-Globin as internal control. Nucleic acid for detecting HBV cccDNA extracted by phenol-chloroform was digested by plasmid-safe ATP dependent DNase which was applied to digest the single strand DNA in rcDNA and ssDNA, then was quantitated by the primers spanning across the nick and SYBR Green Ⅰdye. The specifity of PCR production was confirmed by the sequence analysis and rcDNA comparison. The significance of the difference of HBV cccDNA level between HBeAg（ ＋ ） and HBeAg（ - ） group was analyzed by two group t test. Results The agarose gelelectrophoresis showed the molecular weight of the PCR production was about 350bp. The coincidence rate of PCR production and goal fragement was nearly 99% by sequence analysis. The result of PCR detection of rcDNA group was negative. The positive rate of HBV cccDNA of liver biopsies of HBeAg （ ＋ ） patients detected by this method was 100% , the level of HBV cccDNA in the liver biopsies of HBeAg （ ＋ ） patients was higher than HBeAb（ ＋ ） patients. Conclusions The specificity of the method is provedby agarose electrophoresis , gene sequencing of the PCR product and rcDNA comparison. The quantitative method that use SYBR GreenⅠ dye and β-Globin as internal control is more specific , sensitive and economical,and more suitable for clinical purpose.

关 键 词：B肝炎病毒 乙型 DNA 环状 

分 类 号：R512.6[医药卫生—内科学]