慢性乙型肝炎患者肝组织中HBV cccDNA定量检测方法的建立

肝脏 ›› 2017, Vol. 22 ›› Issue (10): 930-932.

慢性乙型肝炎患者肝组织中HBV cccDNA定量检测方法的建立

李春艳,陈延平,徐光华,冯继红

716000 延安陕西省延安大学附属医院感染病科

收稿日期:2016-12-23 出版日期:2017-10-15 发布日期:2020-06-16
通讯作者: 陈延平,Email:yaypchen@163.com

Received:2016-12-23 Online:2017-10-15 Published:2020-06-16

摘要/Abstract

摘要： 目的建立一种灵敏、特异地检测肝组织中HBV cccDNA的实时荧光定量PCR方法。方法利用HBV cccDNA与rcDNA结构上的差异,设计检测HBV cccDNA的特异性引物及探针,以含HBV基因组全序列的P1.2Ⅱ质粒为检测HBV cccDNA的标准品,建立标准曲线。肝组织标本中提取的DNA分为2等份,1份经质粒安全ATP依赖的DNA酶(PSAD)酶切,作为检测HBV cccDNA的模板,另1份不酶切作为检测肝组织HBV DNA和β-actin的模板,分别进行肝组织HBV DNA和β-actin的定量测定,并以β-actin为参比,对每个细胞的HBV cccDNA和HBV DNA含量进行标准化。结果肝组织HBV tDNA的线性范围为7.41×10⁶～2.66×10¹¹拷贝数/mg(对数值:9.33±1.27),HBV cccDNA的线性范围为7.63×10⁴～9.46×10⁸拷贝数/mg(对数值:7.55±1.08)。 HBV cccDNA含量:13.066±7.387拷贝数/cell,HBV tDNA含量:1 363.9±153.94拷贝数/cell; 同一患者肝组织cccDNA含量与HBV tDNA含量比值为0.96%;肝组织HBV cccDNA与肝组织HBV tDNA呈显著正相关关系(r=0.859,P<0.01)。结论该实时荧光定量PCR法可以灵敏、特异地检测慢性乙型肝炎患者肝组织中的HBV cccDNA载量。

李春艳,陈延平,徐光华,冯继红. 慢性乙型肝炎患者肝组织中HBV cccDNA定量检测方法的建立[J]. 肝脏, 2017, 22(10): 930-932.

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