慢性乙肝患者石蜡包埋肝组织中HBV cccDNA定量检测方法的建立  

Quantification of covalently closed circular DNA of hepatitis B virus in FFPE liver tissues of chronic hepatitis B patients

作　　者：韩佳琪[1] 钟彦伟[1] 任晓强[1] 邹正升[1] 刘树红[1] 刘学恩[2] 赵景民[1] 徐东平[1] 

机构地区：[1]北京大学解放军302医院教学医院病毒性肝炎研究室,北京100039 [2]北京大学基础医学院病原生物学系,北京100191

出　　处：《解放军医学杂志》2011年第5期459-462,共4页Medical Journal of Chinese People's Liberation Army

基　　金：国家"十一五"传染病重大专项课题(2009ZX10005-017;2008ZX10002-011;2009ZX10004-314);北京市自然科学基金重点课题(7091006);首都医学发展研究基金(2009-3057)

摘　　要：目的建立慢性乙肝患者微量石蜡包埋肝组织共价闭合环状DNA(HBV cccDNA)定量检测方法。方法以37例慢性乙肝患者甲醛固定石蜡包埋肝组织为研究对象,提取肝组织HBV DNA,经不降解质粒的ATP依赖的DNA酶(PSAD)消化后,利用滚环扩增加跨缺口实时荧光PCR扩增技术检测肝组织中HBV cccDNA含量,以β-actin为内参照。通过已知浓度的模板DNA进行梯度稀释鉴定该方法的灵敏度,并对该方法进行批内和批间重复性检测。应用该方法分析37例慢性乙肝患者肝组织HBV cccDNA与血清总HBV DNA、肝组织总HBV DNA的关系,以及肝组织HBV cccDNA和HBV DNA与血清HBeAg表达之间的关系。结果成功建立了微量石蜡包埋肝组织HBV cccDNA的定量检测方法,该方法具有较好的特异性、灵敏度和稳定性。HBeAg阳性者肝组织中cccDNA含量明显高于HBeAg阴性者,HBV cccDNA水平与血清总HBV DNA水平(R2=0.48,P=0.042)及肝组织总HBV DNA水平(R2=0.63,P=0.001)呈正相关。结论该方法可特异灵敏地定量检测微量石蜡包埋组织切片中的HBV cccDNA。Objective To establish a method of detecting HBV covalently closed circular DNA（cccDNA） in micro-formalin fixed paraffin imbedding（FFPE） liver biopsy samples.Methods FFPE liver biopsies from 37 patients with chronic hepatitis B were studied.The intrahepatic HBV DNA was extracted and pre-treated with plasmid-safe ATP-dependent DNAse（PSAD）,and then amplified by rolling circular amplification（RCA）.The HBV cccDNA was quantitatively detected by Taqman real-time PCR with primers located on both sides of the gap of HBV DNA.The human β-actin gene served as the internal control.The sensitivity was tested by serially diluting the DNA templates with known concentrations.The repeatability and stability were evaluated with inter-assay and intra-assay.The level of intrahepatic HBV cccDNA,HBV total DNA,serum HBV DNA and ALT were also compared to find the relations between them.Results The quantitative detection method of cccDNA in micro-FFPE liver samples was successfully set up with considerable sensitivity,stability and specificity.The intrahepatic cccDNA level was significantly higher in the HBeAg-positive patients than in the HBeAg-negative patients（P〈0.05）.The intrahepatic HBV cccDNA level was positively correlated with the serum and intra-hepatic HBV DNA level（r=0.539,P=0.001）.Conclusion The assay established by present study is fit for the detection of HBV cccDNA in micro-FFPE liver biopsies.

关 键 词：肝炎病毒 乙型 DNA 环状 核酸扩增技术 

分 类 号：R512.62[医药卫生—内科学]