摘要目的 比较HBV共价闭合环状DNA (Covalently closed circular DNA,cccDNA) Real-time PCR检测方法.方法 选择文献中常用的普通Taq-Man探针及Taq-Man MGB探针两种检测方法,合成引物及探针,制备质粒标准品,提取乙肝患者血清及肝组织标本HBV DNA,分别用质粒安全ATP依赖的DNA酶(Plasmid-safe ATP-dependent DNase,PSAD)酶切,进行敏感性、特异性及重复性检测验证.结果 两种检测方法扩增质粒标准品均有良好线性关系(R20.989和0.976),重复性良好(CV＜4％);检测PSAD酶切前后质粒、血清及肝组织HBV cccDNA均有良好特异性;检测相同浓度样品时普通Taq-Man探针Ct值较Taq-Man MGB探针略低.结论 两种方法均可用于HBV cccDNA检测,本实验所用的普通Taq-Man探针较Taq-Man MGB探针敏感性略高;MGB探针本底更低.实际工作中可根据需求选择适宜的探针.

abstractsObjective To compare two Taq-man Real-time PCR methods for detection of hepatitis B virus covalently closed circular DNA (HBV cccDNA) in serum or liver tissue.Methods Two sets of primers and probes (common Taq-Man probe and MGB Taq-Man probe) were synthesized according to the reference papers,and the sensitivity and specificity of the two methods were compared using prepared plasmid as standard curve,and HBV DNA samples were exlracted from serum and liver tissue samples of hepatitis B patients.The samples were tested with both methods separately before or after the digestion with a Plasmid-Safe ATP-dependent Dnase (PSAD).Results Both of these two kinds of detection methods had a good linear relationship with the prepared plasmid as standard curve (R2 0.989 or 0.976 respectively,CV were within 4％),and obtained good specificity when the HBV DNA samples were tested before or after digestion with PSAD.The common Taq-Man probe had lower Ct value than MGB probe when the samples in the same concentration.Conclusions Both methods can be used for HBV cccDNA detection.The common Taq-Man probe has slightly higher sensitivity than MGB probe,while the MGB probe has lower background than the common Taq-Man probe in our test.One can select the appropriate probe according to the need.