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乙型肝炎病毒cccDNA巢式-实时荧光定量PCR法的建立及应用

许春海 李兆申 代俊英 朱海洋 于健武 吕淑兰

引用本文:
Citation:

乙型肝炎病毒cccDNA巢式-实时荧光定量PCR法的建立及应用

基金项目: 

黑龙江省自然基金资助(D200644); 

详细信息
  • 中图分类号: R512.62

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

Research funding: 

 

  • 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 cccDN...

     

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  • 出版日期:  2011-03-20
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