乙型肝炎病毒cccDNA巢式-实时荧光定量PCR法的建立及应用  

Establishment and application of nested real-time fluorescent quantitative polymerase chain reaction assay for detection of hepatitis B virus covalently closed circular DNA

作　　者：许春海[1] 李兆申[2] 代俊英[1] 朱海洋[1] 于健武[1] 吕淑兰[1] 

机构地区：[1]哈尔滨医科大学第二附属医院,哈尔滨150086 [2]解放军第二军医大学长海医院,上海200433

出　　处：《临床肝胆病杂志》2011年第3期283-285,291,共4页Journal of Clinical Hepatology

基　　金：黑龙江省自然基金资助(D200644)

摘　　要：目的建立检测HBV共价闭合环状(cccDNA)的巢式-荧光定量PCR法,检测外周血单核细胞(PBMC)及骨髓单核细胞(MMNC)中cccDNA。方法根据HBV cccDNA与松弛结构DNA(rcDNA)结构上的差异,设计2对跨缺口的特异性引物及下游的特异性TaqMan探针。根据Mung Bean Nuclease对rcDNA与cccDNA作用的不同,使cccDNA扩增而使rcDNA降解,分别用外引物及内引物进行PCR反应,再用荧光探针进行实时荧光定量PCR,根据阳性参照物,计算出检测标本定量值。结果成功建立了HBV cccDNA巢式-荧光定量PCR的检测方法,线性范围为5.0×102～3.9×107拷贝/ml。用上述方法检测25例慢性乙型肝炎(CHB)及肝硬化血清HBV DNA阳性患者PBMC中cccDNA,7例MMNC中cccDNA,21例健康献血者PBMC cccDNA,骨髓标本中有3例cccDNA阳性,25例外周血标本中有9例cccDNA阳性。结论巢式-荧光定量PCR法可检测乙型肝炎患者PBMC及MMNC中的HBV cccDNA含量。PBMC及MMNC中可检测到HBV cccDNA。Objective To establish a nested real-time quantitative polymerase chain reaction（PCR） assay for detection of hepatitis B virus covalently closed circular DNA（cccDNA）in peripheral blood monocyte（PBMC） and marrow monocyte（MMNC） respectively.Methods Based on the structural differences between HBVcccDNA and HBV rcDNA,two pairs of specific primers spanned the gap of the positive and negative chains and a specific TaqMan probe situated downstream were designed.RcDNA was degraded and cccDNA was processed by Mung Bean Nuclease,cccDNA was amplified by nested real-time quantitative PCR using a pair of outer primers and a pair of inner primers.According to the standard preparation,cccDNA levels of specimen were calculated.Results We established a nested real-time fluorescent quantitative PCR method for HBV cccDNA successfully,and the linear range is from 3.0×102 to 3.9×l08 copies per milliliter.Of the 25 PBMC samples and 7 MMNC samples of the chronic hepatitis B or liver cirrhosis patients,9 PBMC samples and 3 MMNC samples were HBV cccDNA positive,while all of the 21 healthy donator blood PBMC samples were negative.Conclusion The nested real-time fluorescent quantitative PCR method may be applied to detect HBV cccDNA in PBMC and MMNC of patients with HBV.HBV cccDNA can be detected in PBMC and MMNC.

关 键 词：肝炎病毒 乙型 DNA 环状 聚合酶链反应 单核细胞 

分 类 号：R512.62[医药卫生—内科学]